Methods and Compositions for Treating and Preventing HIV

ABSTRACT

Methods and compositions are provided that can be used to vaccinate against and treat HIV. Specifically contemplated are vaccine compositions and methods of using these compositions treat HIV in patients. Aspects of the disclosure relate to an anti-CD40 antibody-HIV antigen fusion protein comprising (i) an anti-CD40 heavy chain (HCD40)-HIV antigen (Ag) fusion protein comprising the formula: HCD40-Ag, wherein Ag is a polypeptide with at least 80% sequence identity to SEQ ID NO:1; and (ii) an anti-CD40 light chain (LCD40).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/563,158, filed Sep. 26, 2017, herebyincorporated by reference in its entirety.

BACKGROUND 1. Field of the Invention

The present invention relates generally to the field of prophylactic andtherapeutic vaccines against an HIV infection. More specifically, theinvention relates to a HIV vaccine comprising an anti-DC receptorantibody or a fragment thereof, e.g. anti-CD40 antibody or a fragmentthereof, to which at least one HIV antigen is fused or conjugated.

2. Description of Related Art

While treatment for HIV/AIDS has become a reality, in the United Statesan average of 50,000 new HIV infections are diagnosed each year in theUnited States and there is an estimated 34 million people living withHIV worldwide.

While a variety of options have been explored, dendritic cells (DCs) areantigen-presenting cells that play a key role in regulatingantigen-specific immunity (Mellman and Steinman 2001), (Banchereau,Briere et al. 2000), (Cella, Sallusto et al. 1997). DCs captureantigens, process them into peptides, and present these to T cells.Therefore delivering antigens directly to DC is a focus area forimproving vaccines. One such example is the development of DC-basedvaccines using ex-vivo antigen-loading of autologous DCs that are thenre-administrated to patients (Banchereau, Schuler-Thurner et al. 2001),(Steinman and Dhodapkar 2001).

Another strategy to improve vaccine efficacy is specific targeting to DCof antigen conjugated to antibodies against internalizing DC-specificreceptors. While first generation polypeptides have been created,improvements are needed to generate more efficacious therapeuticoptions. With the continued epidemic of AIDS throughout the world, thereis still a need for HIV vaccines and treatment methods.

SUMMARY OF THE INVENTION

Methods and compositions are provided that can be used to vaccinateagainst and treat HIV. Specifically contemplated are vaccinecompositions and methods of using these compositions treat or prevent asubject in need thereof from HIV infection. Aspects of the disclosurerelate to an anti-dendritic cell (DC) receptor antibody-HIV antigenfusion protein comprising (i) an anti-DC receptor heavy chain (HDCR)-HIVantigen (Ag) fusion protein comprising the formula: HDCR-Ag, wherein Agis a polypeptide with at least 80% sequence identity to SEQ ID NO:1; and(ii) an anti-DC receptor light chain (LDCR).

In some embodiments, the anti-DC receptor antibody is selected from thegroup consisting of anti-DCIR antibody, anti-LOX-1 antibody,anti-langerin antibody and anti-CD40 antibody.

In a specific embodiment, the anti-DC receptor antibody is an anti-CD40antibody having a heavy chain (HCD40) and a light chain (LCD40).

In some embodiments, the fusion protein further comprises one or morepeptide linkers (PL). In some embodiments, the fusion protein comprises:(i) HCD40-PL-Ag; and (ii) LCD40. In some embodiments, the fusion proteinfurther comprises one or more joining sites (JS), wherein the joiningsite comprises alanine and serine residues. In some embodiments, thejoining site consists of alanine and serine residues. In someembodiments, the fusion protein comprises: (i) HDCR-JS-Ag-JS; and (ii)LDCR. In some embodiments, the fusion protein comprises (i)HCD40-JS-PL-JS-Ag-JS; and (ii) LCD40. In some embodiments, the peptidelinker comprises a polypeptide with at least 80% sequence identity toSEQ ID NO:2. In some embodiments, the peptide linker comprises apolypeptide with at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity(or any derivable range therein) to SEQ ID NO:2, or a fragment of atleast 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, or 26 (or any derivable range therein) contiguous amino acids ofSEQ ID NO:2.

In some embodiments, PL-JS-Ag-JS comprises a polypeptide with at least80% identity to SEQ ID NO:3. In some embodiments, PL-JS-Ag-JS comprisesa polypeptide with at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity(or any derivable range therein) to SEQ ID NO:3, or a fragment of atleast 100, 200, 300, 400, 500, 600, 610, 615, 620, 625, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, or661 (or any derivable range therein) contiguous amino acids of SEQ IDNO:3.

In some embodiments, the anti-DC receptor antibody, e.g. the anti-CD40antibody is a human or humanized antibody. In some embodiments, theanti-DC receptor antibody, e.g. the anti-CD40 antibody, comprises humanIgG4 heavy chain constant region. In some embodiments, the human IgG4heavy chain constant region comprises one or both of S241P and L248Esubstitutions according to Kabat numbering.

In some embodiments, the HCD40 comprises the complementarity determiningregions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequenceGFTFSDYYMY (SEQ ID NO:10), the CDR2H having the amino acid sequenceYINSGGGSTYYPDTVKG (SEQ ID NO.:11), and the CDR3H having the amino acidsequence RGLPFHAMDY (SEQ ID NO.:12). In some embodiments, the LCD40 iscomprises the complementarity determining regions CDR1L, CDR2L andCDR3L, the CDR1L having the amino acid sequence SASQGISNYLN (SEQ IDNO.:13) the CDR2L having the amino acid sequence YTSILHS (SEQ ID NO.:14)and the CDR3L having the amino acid sequence QQFNKLPPT (SEQ ID NO.:15).In some embodiments, the anti-DC receptor antibody comprises a HCD40comprising the complementarity determining regions CDR1H, CDR2H andCDR3H, the CDR1H having the amino acid sequence GFTFSDYYMY (SEQ IDNO.:10), the CDR2H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQID NO.:11), and the CDR3H having the amino acid sequence RGLPFHAMDY (SEQID NO.:12) and a LCD40 comprising the complementarity determiningregions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequenceSASQGISNYLN (SEQ ID NO.:13) the CDR2L having the amino acid sequenceYTSILHS (SEQ ID NO.:14) and the CDR3L having the amino acid sequenceQQFNKLPPT (SEQ ID NO.:15).

In some embodiments, the HCD40 comprises the complementarity determiningregions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequenceGYSFTGYYMH (SEQ ID NO.:18), the CDR2H having the amino acid sequenceRINPYNGATSYNQNFKD (SEQ ID NO.:19), and the CDR3H having the amino acidsequence EDYVY (SEQ ID NO.:20). In some embodiments, the LCD40 iscomprises the complementarity determining regions CDR1L, CDR2L andCDR3L, the CDR1L having the amino acid sequence RSSQSLVHSNGNTYLH (SEQ IDNO.:21) the CDR2L having the amino acid sequence KVSNRFS (SEQ ID NO.:22)and the CDR3L having the amino acid sequence SQSTHVPWT (SEQ ID NO.:23).In some embodiments, the anti-DC receptor antibody comprises a HCD40comprising the complementarity determining regions CDR1H, CDR2H andCDR3H, the CDR1H having the amino acid sequence GYSFTGYYMH (SEQ IDNO.:18), the CDR2H having the amino acid sequence RINPYNGATSYNQNFKD (SEQID NO.:19), and the CDR3H having the amino acid sequence EDYVY (SEQ IDNO.:20) and a LCD40 comprising the complementarity determining regionsCDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequenceRSSQSLVHSNGNTYLH (SEQ ID NO.:21) the CDR2L having the amino acidsequence KVSNRFS (SEQ ID NO.:22) and the CDR3L having the amino acidsequence SQSTHVPWT (SEQ ID NO.:23).

In some embodiments, the HCD40 comprises the complementarity determiningregions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequenceGYTFTDYVLH (SEQ ID NO.:26), the CDR2H having the amino acid sequenceYINPYNDGTKYNEKFKG (SEQ ID NO.:27), and the CDR3H having the amino acidsequence GYPAYSGYAMDY (SEQ ID NO.:28). In some embodiments, the LCD40 iscomprises the complementarity determining regions CDR1L, CDR2L andCDR3L, the CDR1L having the amino acid sequence RASQDISNYLN (SEQ IDNO.:29) the CDR2L having the amino acid sequence YTSRLHS (SEQ ID NO.:30)and the CDR3L having the amino acid sequence HHGNTLPWT (SEQ ID NO.:31).In some embodiments, the anti-DC receptor antibody comprises a HCD40comprising the complementarity determining regions CDR1H, CDR2H andCDR3H, the CDR1H having the amino acid sequence GYTFTDYVLH (SEQ IDNO.:26), the CDR2H having the amino acid sequence YINPYNDGTKYNEKFKG (SEQID NO.:27), and the CDR3H having the amino acid sequence GYPAYSGYAMDY(SEQ ID NO.:28) and a LCD40 comprising the complementarity determiningregions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequenceRASQDISNYLN (SEQ ID NO.:29) the CDR2L having the amino acid sequenceYTSRLHS (SEQ ID NO.:30) and the CDR3L having the amino acid sequenceHHGNTLPWT (SEQ ID NO.:31).

In some embodiments, the HCD40 comprises the complementarity determiningregions CDR1H, CDR2H and CDR3H, the CDR1H having an amino acid sequencethat is at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to one of SEQID NOS: 10, 18, and 26, the CDR2H having an amino acid sequence that isat least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to one of SEQ IDNOS:11, 19, and 27, and the CDR3H having an amino acid sequence that isat least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to one of SEQ IDNOS:12, 20, and 28. In some embodiments, the LCD40 is comprises thecomplementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1Lhaving an amino acid sequence that is at least 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99% identical to one of SEQ ID NOS: 13, 21, and 29, the CDR2L havingan amino acid sequence that is at least 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to one of SEQ ID NOS: 14, 22, and 30, and the CDR3L having anamino acid sequence that is at least 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to one of SEQ ID NOS:15, 23, and 31.

In some embodiments, the HCD40 comprises a polypeptide with at least 80%sequence identity to SEQ ID NO:4. In some embodiments, the HCD40comprises a polypeptide with at least 70, 75, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequenceidentity (or any derivable range therein) to SEQ ID NO:4. In someembodiments, the LCD40 comprises a polypeptide with at least 80%sequence identity to SEQ ID NO:5. In some embodiments, the LCD40comprises a polypeptide with at least 70, 75, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequenceidentity (or any derivable range therein) to SEQ ID NO:5.

In some embodiments, the fusion protein comprises: (i) a HCD40-Ag fusionprotein comprising an amino acid sequence with at least 80% sequenceidentity to SEQ ID NO:6; and (ii) a LCD40 comprising an amino acidsequence with at least 80% identity to SEQ ID NO:5.

Throughout the disclosure, LCD40 refers to a light chain of an anti-CD40antibody. Likewise, HCD40 refers to a heavy chain of an anti-CD40antibody.

Further aspects of the disclosure relate to a polynucleotide encodingfor a fusion protein of the disclosure. In some embodiments, thepolynucleotide comprise a nucleotide sequence that is at least 80%identical (or any derivable range therein) to SEQ ID NO: 7. In someembodiments, the polynucleotide comprise a nucleotide sequence that isat least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% identical (or any derivable rangetherein) to SEQ ID NO: 7. In some embodiments, the polynucleotidecomprise a nucleotide sequence that is at least 80% identical (or anyderivable range therein) to SEQ ID NO: 8. In some embodiments, thepolynucleotide comprise a nucleotide sequence that is at least 70, 75,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical (or any derivable range therein) to SEQ ID NO:8.

Further aspects relate to an expression vector comprising one or morepolynucleotides of the disclosure. Yet further aspects relate to a hostcell comprising one or more polynucleotides, expression vectors, orfusion proteins of the disclosure. In some embodiments, the host cellsis a mammalian cell. In some embodiments, the mammalian cells compriseChinese hamster ovary cells. Further aspects relate to a fusion proteinisolated from a host cell of the disclosure.

Expression vectors may be constructed with diverse protein codingsequence e.g., fused in-frame to the H chain coding sequence. Forexample, HIV antigens may be expressed subsequently as Ab.Ag_(x), whichin the context of this invention, can have utility derived from usingthe anti-CD40 V-region sequence to bring the antigen directly to thesurface of the antigen presenting cell bearing CD40. This permitsinternalization of e.g., antigen and ensuing initiation of therapeuticor protective action (e.g., via initiation of a potent immune response).

In some cases, particular amino acid sequences corresponding to anti-DCreceptor monoclonal antibodies that are desirable components (in thecontext of e.g., humanized recombinant antibodies) of therapeutic orprotective products. The following are such sequences in the context ofchimeric mouse V region (underlined) human C region recombinantantibodies. These mouse V regions can be readily humanized, i.e., thecombining regions grafted onto human V region framework sequences, byanyone well practiced in this art. Furthermore, the sequences can alsobe expressed in the context of fusion proteins that preserve antibodyfunctionality, but add e.g., antigen for desired therapeuticapplications.

Further aspects relate to a vaccine comprising a fusion protein of thedisclosure and a pharmaceutically acceptable vehicle.

Further aspects relate to a method for treating or preventing from HIVinfection in a subject comprising administering a fusion protein of thedisclosure to the subject. Yet further aspects relate to a method foreliciting and/or enhancing B-cell and/or T-cell response against HIV, ina subject in need thereof, comprising administering to said subject inneed thereof, a fusion protein or vaccine of the disclosure. Furtheraspects relate to a method for inducing IgG binding antibody responsesto V1V2 region antigens in a subject in need thereof, the methodcomprising administering the fusion protein of the disclosure or thevaccine composition of the disclosure.

In some embodiments, the method further comprises administration of animmunostimulant. In some embodiments, the immunostimulant isadministered sequentially or concomitantly to the vaccine composition.In some embodiments, the immunostimulant is mixed with the vaccinecomposition extemporaneously prior to injection of the vaccinecomposition to the subject.

Vaccine compositions may also contain one or more adjuvants.Additionally, the methods of the disclosure may also comprise theadministration of one or more adjuvants. The adjuvants may be attachedor conjugated directly or indirectly to one or more of the vaccinecomponents, such as an antigen or antibody. In other embodiments theadjuvants may be provided or administered separately from the vaccinecomposition. In certain embodiments the adjuvant is poly ICLC, CpG, LPS,Immunoquid, PLA, GLA or cytokine adjuvants such as IFNα. In otherembodiments the adjuvant may be a toll-like receptor agonist (TLR).Examples of TLR agonists that may be used comprise TLR1 agonist, TLR2agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7agonist, TLR8 agonist or TLR9 agonist. In certain embodiments, a vaccinecomposition specifically does not contain PLA as an adjuvant. In someembodiments, the immunostimulant comprises a TLR agonist. In someembodiments, the TLR agonist comprises poly ICLC.

In some embodiments, the method excludes administration of a TLRagonist.

In some embodiments, an HIV vaccine comprises a population of dendriticcells (DC) activated with an antibody-antigen fusion protein (Ab.Ag) oran antibody-antigen complex (Ab:Ag) as described for anti-CD40 vaccines.In yet other embodiments a first HIV vaccine of the disclosure, e.g.anti-CD40 vaccine is combined with a second HIV vaccine. The second HIVvaccine may be selected from the group consisting of an attenuatedrecombinant virus or viral-vector based, Virus-like-particle (VLP)vaccine, DNA vaccine (naked or not), RNA-based vector vaccine,protein-based vaccine and a DC-targeting vaccine. In certainembodiments, the attenuated recombinant virus may be an attenuatedrecombinant poxvirus. In certain embodiments, the second vaccine isDNA-HIV-PT123.

In some embodiments, the method further comprises administration of anadditional vaccine. In some embodiments, the additional vaccine isadministered as a priming vaccine. In some embodiments, the additionalvaccine comprises a vaccinia virus vaccine. In some embodiments, theadditional vaccine comprises one or more of a gag, pol, and nef peptide.

In some embodiments, the administration comprises intradermal,intramuscular, or subcutaneous administration.

In some embodiments, the additional vaccine comprises an attenuatedrecombinant poxvirus. In some embodiments, the additional vaccine isselected from the group consisting of NYVAC, ALVAC and MVA virus.Specifically, the NYVAC virus may be a NYVAC-KC virus.

In some embodiments, the HIV vaccine, e.g. anti-CD40 HIV vaccine, isused in a method for potentiating an immune response to at least one HIVepitope comprising administering to a patient such HIV vaccine asdescribed herein. In some embodiments, such HIV vaccine is used toprevent healthy subject to be infected by HIV, comprising administeringsuch HIV vaccine of the present disclosure, e.g. HIV anti-CD40 vaccineto a healthy subject, not infected by HIV (preventive treatment). Inother embodiments, the HIV vaccine of the present disclosure is used ina method of treating a patient in the early stages of an HIV infectioncomprising administering to a patient an anti-CD40 HIV vaccine.

It is contemplated that at least one HIV antigen elicits at least one ofa humoral and/or a cellular immune response in a host, preferably ahuman patient or a primate.

Further aspects of the disclosure relate to a method for making ananti-DC receptor antibody-HIV antigen fusion protein, the methodcomprising expressing and polynucleotide of the disclosure or anexpression vector of the disclosure in a host cell and purifying thefusion protein from the host cell. In some embodiments, the host cell isa CHO cell. In some embodiments, at least 1.5 mg/mL of the fusionprotein is purified. In some embodiments, at least 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9. 3.0, 3.2, 3.4, 3.6, 3.8, or 4 mg/mL (or anyderivable range therein) is purified from the host cell.

The preparation of HIV vaccine as the active immunogenic ingredient, maybe prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinfection can also be prepared. The preparation may be emulsified,encapsulated in liposomes. The active immunogenic ingredients are oftenmixed with carriers which are pharmaceutically acceptable and compatiblewith the active ingredient.

The term “pharmaceutically acceptable carrier” refers to a carrier thatdoes not cause an allergic reaction or other untoward effect in subjectsto whom it is administered. Suitable pharmaceutically acceptablecarriers include, for example, one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine can containminor amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, and/or adjuvants which enhance theeffectiveness of the vaccine. Examples of adjuvants that may beeffective include but are not limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, whichcontains three components extracted from bacteria, monophosphoryl lipidA, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion. Other examples of adjuvants include DDA(dimethyldioctadecylammonium bromide), Freund's complete and incompleteadjuvants and QuilA. In addition, immune modulating substances such aslymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma]inducers such as poly I:C or poly ICLC (Hiltonol) can be used incombination with adjuvants described herein.

Vaccines may include an effective amount of the antibody-antigen fusionprotein (Ab.Ag) or the antibody-antigen complex (Ab:Ag), dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be referred to as inocula. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. The compositions of the presentinvention may include classic pharmaceutical preparations. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

Administration of vaccines according to the present invention will bevia any common route so long as the target tissue is available via thatroute in order to maximize the delivery of antigen to a site for maximum(or in some cases minimum) immune response. Administration willgenerally be by orthotopic, intradermal, mucosally, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Other areas fordelivery include: oral, nasal, buccal, rectal, vaginal or topical.Vaccines of the invention are preferably administered parenterally, byinjection, for example, either subcutaneously or intramuscularly.

Vaccines may be administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective. The quantity to be administered depends onthe subject to be treated, including, e.g., capacity of the subject'simmune system to synthesize antibodies, and the degree of protection ortreatment desired. Suitable dosage ranges are of the order of severalhundred micrograms active ingredient per vaccination with a range fromabout 0.1 mg to 1000 mg, such as in the range from about 1 mg to 300 mg,or in the range from about 10 mg to 50 mg. Suitable regiments forinitial administration and booster shots are also variable but aretypified by an initial administration followed by subsequentinoculations or other administrations. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and may be peculiar to each subject. It will be apparent tothose of skill in the art that the therapeutically effective amount ofnucleic acid molecule or fusion polypeptides of this invention willdepend, inter alia, upon the administration schedule, the unit dose ofantigen administered, whether the antibody-antigen fusion protein(Ab.Ag) or the antibody-antigen complex (Ab:Ag) is administered incombination with other therapeutic agents, the immune status and healthof the recipient, and the therapeutic activity of the particular theAb.Ag or the Ab:Ag.

A vaccine may be given in a single dose schedule or in a multiple doseschedule. A multiple dose schedule is one in which a primary course ofvaccination may include, e.g., 1-10 separate doses, followed by otherdoses given at subsequent time intervals required to maintain and orreinforce the immune response, for example, at 1-4 months for a seconddose, and if needed, a subsequent dose(s) after several months. Periodicboosters at intervals of 1-5 years, usually 3 years, are desirable tomaintain the desired levels of protective immunity. The course of theimmunization can be followed by in vitro proliferation assays ofperipheral blood lymphocytes (PBLs) co-cultured with gp140 antigen, andby measuring the levels of IFN-[gamma] released from the primedlymphocytes. The assays may be performed using conventional labels, suchas radionucleotides, enzymes, fluorescent labels and the like. Thesetechniques are known to one skilled in the art and can be found in U.S.Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant portionsincorporated by reference.

A vaccine may be provided in one or more “unit doses”. Unit dose isdefined as containing a predetermined-quantity of the vaccine calculatedto produce the desired responses in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, and the particular route and formulation, are within theskill of those in the clinical arts. The subject to be treated may alsobe evaluated, in particular, the state of the subject's immune systemand the protection desired. A unit dose need not be administered as asingle injection but may include continuous infusion over a set periodof time. The amount of vaccine delivered can vary from about 0.001 toabout 0.05 mg/kg body weight, for example between 0.1 to 5 mg persubject. Thus, in particular embodiments, 0.3 mg, 0.4 mg, 0.5 mg, 0.6mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3.0 mg, 3.1 mg, 3.2 mg, 3.3mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4.0 mg, 4.1 mg, 4.2mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, or 5.0 mg(or any derivable range therein), of the antibody-HIV antigen fusionprotein in the vaccine and/or additional vaccine may be delivered to anindividual in vivo. In some embodiments, the amount of vaccine deliveredcan be at least, at most, or exactly about 0.001 mg/kg, 0.002 mg/kg,0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.012 mg/kg, 0.014 mg/kg, 0.016 mg/kg,0.018 mg/kg, 0.02 mg/kg, 0.022 mg/kg, 0.024 mg/kg, 0.026 mg/kg, 0.028mg/kg, 0.03 mg/kg, 0.032 mg/kg, 0.034 mg/kg, 0.036 mg/kg, 0.038 mg/kg,0.04 mg/kg, 0.042 mg/kg, 0.044 mg/kg, 0.046 mg/kg, 0.048 mg/kg, 0.05mg/kg, 0.052 mg/kg, 0.054 mg/kg, 0.056 mg/kg, 0.058 mg/kg, 0.06 mg/kg,0.062 mg/kg, 0.064 mg/kg, 0.066 mg/kg, 0.068 mg/kg, or 0.07 mg/kg (orany derivable range therein), of the antibody-HIV antigen fusion proteinin the vaccine and/or additional vaccine may be delivered to anindividual in vivo. The dosage of vaccine to be administered depends toa great extent on the weight and physical condition of the subject beingtreated as well as the route of administration and the frequency oftreatment.

In another aspect, embodiments relate to a combined HIV vaccinecomprising a first HIV vaccine as described above and a second HIVvaccine. Therefore, in some methods a patient may be administered bothan HIV vaccine composition discussed above, such as a dendritic-cellantibody attached to an HIV antigen, and a recombinant attenuatedpoxvirus that encodes for at least one HIV antigen. It is contemplatedthat in some embodiments the recombinant attenuated poxvirus isadministered after the first HIV vaccine composition has beenadministered. It is further contemplated that the HIV antigens providedto the patient in the first and second HIV vaccines may be the same orthey may be different. It is also contemplated that the administrationof the first and second vaccines can be reversed such that the secondvaccine is administered first and the first vaccine is administeredsecond. It additionally contemplated that the first and second vacccinesbe administered at the same time. In instances when the first and secondvaccines are not administered at the same time it is contemplated thatthe vaccines may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days apart or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51 or 52 weeks apart or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84 or 96 monthsapart or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20 years apart (or any derivable range therein. Furthermore, anyone vaccine (either the fusion protein of the disclosure or theadditional vaccine) may be administered at least, at most, or exactly 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or any derivable range therein)throught one course of treatment, such as a prescribed course oftreatment by a medical professional. When the vaccine is administeredmore than once, it may be administered at least, at most, or exactly 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days apart or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 weeks apart or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 36, 48, 60, 72, 84 or 96 months apart or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 years apart (or anyderivable range therein).

In one embodiment, the second HIV vaccine is selected from the groupconsisting of an attenuated recombinant virus or viral-vector based,Virus-like-particle (VLP) vaccine, DNA vaccine (naked or not), RNA-basedvector vaccine, protein-based vaccine and a DC-targeting vaccine. Incertain embodiments, the attenuated recombinant virus may be anattenuated recombinant poxvirus.

Further aspects relate to a kit comprising a fusion protein of thedisclosure, a polynucleotide of the disclosure, an expression vector ofthe disclosure, or a host cell of the disclosure, and; optionally,instructions for use of the kit. The kit may be used to perform themethods described herein. In some embodiments, the kit is for elicitinga T cell response and/or a B cell response in a subject; wherein the kitcomprises the fusion protein of the disclosure or the vaccine of thedisclosure.

As used herein, the term “attenuated recombinant virus” refers to avirus that has been genetically altered by modern molecular biologicalmethods, e. g. restriction endonuclease and ligase treatment, andrendered less virulent than wild type, typically by deletion of specificgenes or by serial passage in a non-natural host cell line or at coldtemperatures.

In one particular embodiment, the attenuated recombinant virus is anattenuated recombinant poxvirus.

“Poxviruses” are large, enveloped viruses with double-stranded DNA thatis covalently closed at the ends. Pox viruses replicate entirely in thecytoplasm, establishing discrete centers of viral synthesis. Their useas vaccines has been known since the early 1980's (see, e. g. Panicali,et al., 1983).

In one particular embodiment, the attenuated recombinant poxvirus isselected from the group consisting of NYVAC, ALVAC and MVA virus. In oneembodiment, the NYVAC virus is a NYVAC-KC virus. In other embodiments,the second HIV vaccine is DNA-HIV-PT123.

In some embodiments, the mAb is humanized (i.e., converted to a sequencewhich retains the original key residues crucial for receptor binding,but has variable region framework and constant region sequences that aretypically found in human antibodies).

As used herein, the term “vaccine” is intended to mean a compositionwhich can be administered to humans or to animals in order to induce animmune response; this immune response can result in a production ofantibodies or simply in the activation of certain cells, in particularantigen-presenting cells, T lymphocytes and B lymphocytes. In certainembodiments the vaccine is capable of producing an immune response thatleads to the production of neutralizing antibodies in the patient withrespect to the antigen provided in the vaccine. The vaccine can be acomposition for prophylactic purposes or for therapeutic purposes, orboth.

As used herein, the term “antigen” refers to any antigen that can beused in a vaccine, whether it involves a whole microorganism or aportion thereof, and various types: (e.g., peptide, protein,glycoprotein, polysaccharide, glycolipid, lipopeptide, etc). Thus, theterm “antigen” refers to a molecule that can initiate a humoral and/orcellular immune response in a recipient of the antigen. The antigen isusually a molecule that causes a disease for which a vaccination wouldbe advantageous treatment. Within the context of the invention, theantigens are human immunodeficiency virus (HIV) antigens; the term“antigen” also comprises the polynucleotides, the sequences of which arechosen so as to encode the antigens whose expression by the individualsto which the polynucleotides are administered is desired, in the case ofthe immunization technique referred to as DNA immunization.

As used herein, the term “antibodies” refers to immunoglobulins, whethernatural or partially or wholly produced artificially, e.g. recombinant.An antibody may be monoclonal or polyclonal. The antibody may, in somecases, be a member of one, or a combination immunoglobulin classes,including: IgG, IgM, IgA, IgD, and IgE.

As used herein, the term “antibody or fragment thereof,” includes wholeantibodies or fragments of an antibody, e.g., Fv, Fab, Fab′, F(ab′)2,Fc, and single chain Fv fragments (ScFv) or any biologically effectivefragments of an immunoglobulins that binds specifically to, e.g., CD40.Antibodies from human origin or humanized antibodies have lowered or noimmunogenicity in humans and have a lower number or no immunogenicepitopes compared to non-human antibodies. Antibodies and theirfragments will generally be selected to have a reduced level or noantigenicity in humans. A polypeptide that has one or more CDRs from amonoclonal antibody and that may have at least as good as a bindingspecificity and/or affinity of a monoclonal antibody may be referred toas an “antibody fragment” or a polypeptide comprises an antibodyfragment.

Typically, the term “antibody or fragment thereof” describes arecombinant antibody system that has been engineered to provide a targetspecific antibody. The monoclonal antibody made using standard hybridomatechniques, recombinant antibody display, humanized monoclonalantibodies and the like. The antibody can be used to, e.g., target (viaone primary recombinant antibody against an internalizing receptor,e.g., a human dendritic cell receptor such as CD40) one or severalantigens and/or one adjuvant to dendritic cells. Any embodimentdiscussed in the context of an antibody may be implemented in thecontext of an antibody fragment, including a polypeptide comprising oneor more CDRs from an antibody, more specifically an antibody comprisingthe CDRs 1, 2 and 3 of the heavy chain variable region of a specificantibody and the CDRs 1, 2 and 3 of the light chain variable region ofthe same specific antibody.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and other fragments that exhibit immunological bindingproperties of the parent monoclonal antibody molecule.

As used herein, the term “antigen-binding site” or “binding portion”refers to the part of the immunoglobulin molecule that participates inantigen binding. The antigen binding site is formed by amino acidresidues of the N-terminal variable (“V”) regions of the heavy (“H”) andlight (“L”) chains. Three highly divergent stretches within the Vregions of the heavy and light chains are referred to as “hypervariableregions” which are interposed between more conserved flanking stretchesknown as “framework regions” (FRs). As used herein, the term “FR” refersto amino acid sequences which are found naturally between and adjacentto hypervariable regions in immunoglobulins. In an antibody molecule,the three hypervariable regions of a light chain and the threehypervariable regions of a heavy chain are disposed relative to eachother in three dimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions” or “CDRs”.

As used herein, the term “humanized” antibody refers to those moleculescomprising an antigen-binding site derived from a non-humanimmunoglobulin have been described, including chimeric antibodies havingrodent V regions and their associated CDRs fused to human constantdomains, rodent CDRs grafted into a human supporting FR prior to fusionwith an appropriate human antibody constant domain, and rodent CDRssupported by recombinantly veneered rodent FRs. These “humanized”molecules are designed to minimize unwanted immunological responsetoward rodent antihuman antibody molecules, which limits the durationand effectiveness of therapeutic applications of those moieties in humanrecipients.

The terms “adjuvant” or “immunoadjuvant” may be used interchangeably andrefer to a substance that enhances, augments or potentiates the host'simmune response to an antigen, e.g., an antigen that is part of avaccine. Non-limiting examples of some commonly used vaccine adjuvantsinclude insoluble aluminum compounds, calcium phosphate, liposomes,Virosomes™, ISCOMS®, microparticles (e.g., PLG), emulsions (e.g., MF59,Montanides), virus-like particles & viral vectors. PolyICLC (a syntheticcomplex of carboxymethylcellulose, polyinosinic-polycytidylic acid, andpoly-L-lysine double-stranded RNA), which is a TLR3 agonist, is used asan adjuvant in the present invention. It will be understood that otherTLR agonists may also be used (e.g. TLR4 agonists, TLR5 agonists, TLR7agonists, TLR9 agonists; also as described in PCT publication numberWO/2012/021834 or WO/2012/122396, the contents of which are incorporatedherein by reference), or any combinations or modifications thereof.

As used herein, the term “conjugate” refers to any substance formed fromthe joining together of two parts. Representative conjugates inaccordance with the present invention include those formed by joiningtogether of the antigen with the antibody and/or the adjuvant. The term“conjugation” refers to the process of forming the conjugate and isusually done by physical coupling, e.g. covalent binding, co-ordinationcovalent, or secondary binding forces, e.g. Van der Waals bondingforces. The process of linking the antigen to the antibody and/or to theadjuvant can also be done via a non-covalent association such as adockerin-cohesin association (as described in U.S. Patent PublicationNo. 20100135994, Banchereau et al. relevant portions incorporated hereinby reference) or by a direct chemical linkage by forming a peptide orchemical bond.

As used herein, “Dendritic Cells” (DCs) refers to any member of adiverse population of morphologically similar cell types found inlymphoid or non-lymphoid tissues. These cells are characterized by theirdistinctive morphology, high levels of surface MHC-class II expression(Steinman, et al., 1991); incorporated herein by reference for itsdescription of such cells). These cells can be isolated from a number oftissue sources, and conveniently, from peripheral blood, as describedherein.

As used herein, the term “HIV” refers to the human immunodeficiencyvirus. HIV includes, without limitation, HIV-1. HIV may be either of thetwo known types of HIV, i.e., HIV-1 or HIV-2. The HIV-1 virus mayrepresent any of the known major subtypes or clades (e.g., Classes A, B,C, D, E, F, G, J, and H) or outlying subtype (Group O). Also encompassedare other HIV-1 subtypes or clades that may be isolated.

Methods are provided for preventing or treating an HIV infectioncomprising administering to a subject in need thereof an HIV vaccine ora combination of vaccines as discussed above or herein. In certainembodiments, there are methods for inducing an immune response to atleast one HIV epitope comprising administering to a subject in needthereof an HIV vaccine or a combination of vaccines as discussed aboveor herein. Other methods are provided for potentiating an immuneresponse to at least one HIV epitope comprising administering to asubject in need thereof an HIV vaccine or a combination of vaccines asdiscussed above or herein.

In some embodiments, there are methods of preventing a subject frombeing infected by HIV comprising administering to a subject, for examplea healthy subject diagnosed as non-infected by HIV, an HIV vaccine orcombination of vaccines as discussed above or herein.

In further embodiments there are methods of treating a patient in theearly stages of an HIV infection comprising administering to the patientan HIV vaccine or a combination of vaccines as discussed above orherein.

In some cases, there are methods of treating a patient comprisingadministering to the patient an HIV vaccine or a combination of vaccinesas discussed above or herein.

Methods may involve a patient tested for an HIV infection, a patientdetermined to be infected with HIV, a patient with symptoms of earlystage HIV infection, a patient who is at risk for HIV infection, apatient whose HIV infection status is known, or a patient previouslytreated for HIV infection. In certain embodiments, the patient is apatient who is pregnant. In other embodiments, a patient is a pediatricpatient whose mother was infected with HIV. In other embodiments, thepatient is a pediatric patient above the age of 13 years old. In certainembodiments, the patient has been exposed to HIV.

In some embodiments, methods further comprise testing the patient forHIV infection or diagnosing a patient with HIV infection. Additionalmethods may also involve treating a patient also with other HIVtreatments such as HIV/AIDS small molecule treatments.

In certain aspects, methods further comprise generating a neutralizingantibody. In certain instances, said method comprises administering anHIV vaccine composition of the present disclosure.

Any embodiment discussed in the context of an antibody may beimplemented in any method embodiment discussed herein.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-H. Serum Env gp140-specific IgG responses elicited by αLOX-1.Envgp140 and αCD40.Env gp140 fusion protein administration. Total bindingIgG antibody levels against Clade C gp140 (Vaccine Strain, ZM96) (A),Group M gp140 (Consensus) (B), Clade C gp140 (Consensus) (C), Clade Bgp140 (Consensus) (D), Clade B V1-V2 Env (gp70 B.CaseA V1V2) (E), andClade C V1-V2 (C.1086C V1V2 Tags) (F), Clade AE V1-V2 (AE.A244 V1V2Tags) (G), Clade AE V2 (AE.A244 V2 Tags) (H) induced by the differentvaccination groups are shown. Individual serum samples were obtained atweeks 6, 14, 26, and 32 from each macaque (n=6 per group) immunized asper G1 N2[LpN]2, G2 N2[CpN]2, G3 N2Lp2, G4 N2Cp2, and G5 N2C2 (Table 1).Binding antibodies were measured by BAMA as indicated in Materials andMethods. The magnitudes of the antibody responses are expressed as theAUC from serial dilutions of plasma. Each dot represents the value forone individual macaque. Weeks of sampling are indicated above each frameand groups are defined below each frame. Response rates for each groupare given above each dataset. The mid-line of the box denotes themedian, and the ends of the box denote the 25^(th) and 75^(th)percentiles. The whiskers that extend from the top and bottom of the boxextend to the most extreme data points that are no more than 1.5 timesthe interquartile range (i.e., the height of the box) or if no valuemeets this criterion, to the data extremes. Table 2 shows statisticalanalysis of paired comparisons of serum IgG responses against variousEnv antigens, including those exemplified in this figure.

FIG. 2. Serum Env gp140-specific IgA responses elicited by αLOX-1.Envgp140 and αCD40.Env gp140 fusion protein administration. Total bindingIgA antibody levels against Clade C gp140 (Vaccine Strain, ZM96) inducedby the different vaccination groups are shown. Individual serum sampleswere obtained at weeks 6, 14, 26, and 32 from each macaque (n=6 pergroup) immunized as per G1 N2[LpN]2, G2 N2[CpN]2, G3 N2Lp2, G4 N2Cp2,and G5 N2C2 (Table 1). Binding antibodies were measured as indicated inMaterials and Methods. Each panel shows anti-Env IgA binding units (MFI)of individual NHPs. Plotting details are as described in FIG. 1, exceptIgA levels were log 10-transformed.

FIG. 3. Env gp120 epitope-specific IgG responses elicited by αLOX-1.Envgp140 and αCD40.Env gp140 fusion protein administration. A gp160 peptidearray was probed with week 0 (for baseline) and week 26 (at peakresponse) plasma from 17 animals representing all 5 immunization groups,selected based on higher binding (per group) to ZM96 gp140 and/or 1086V1V2 Tags. Three animals each were tested from Groups 1, 3, and 5, andfour animals each were tested from Group 2 and 4. Mean binding valuesfor each gp120 peptide are shown for group 1 to 5 (G1-G5). Data fromvaccine strains for clade B (MN), clade C (TV1, 1086C and ZM651), andclade AE (A244 and TH023) are combined with the consensus of each torepresent clade B, C, and AE, respectively. Epitope regions identifiedin the study are indicated with texts over horizontal bars in plots.Summary data for each epitope shown here are presented in Table 3.

FIG. 4A-B. Serum neutralizing antibody responses elicited by αLOX-1.Envgp140 and αCD40.Env gp140 fusion protein administration. (A)Neutralizing antibodies against HIV-1 were measured as a function ofreduction in Tat-regulated reporter gene expression in TZM-bl cellsinfected with HIV-1 isolate MW965. Neutralizing antibody titer valuesare ID50. Samples were tested from sera collected at weeks −4, 14, 26,and 32. Groups are indicated beside the graph. Each dot represents thevalue for one individual macaque. (B) The graph shows the area under MBcurves (Material and Methods) used to summarize overall breadth andmagnitude of neutralizing antibody activities against A3R5 virus foreach individual time point. Samples were tested from sera collected atweeks −4, 14, 26, and 32. Groups are indicated below the graph. Table 4shows analysis of paired comparisons of ID50 values for isolates MW965and Th023.6.

FIG. 5. ADCC-mediated antibody responses elicited by αLOX-1.Env gp140and αCD40.Env gp140 fusion protein administration. ADCC-mediating serumantibodies were measured by the GranToxLux assay that measures percentgranzyme B activity against HIV-Env protein-coated cells. Response ratesare given at the top of each graph column.

FIG. 6. Blood HIV-1 antigen-specific T cell responses elicited byαLOX-1.Env gp140 and αCD40.Env gp140 fusion protein administration.PBMCs were analyzed by IFNγ ELISPOT for responses to three Env gp140peptide pools. Sample collection times are shown in weeks below thegraph. Dots are results for individual animals and the box plotsrepresent the distribution of values with the bars indicating the medianvalues for the group. No significant responses were detected againstnon-Env HIV peptides (not shown). Statistical analysis of blood HIV-1antigen-specific T cell responses elicited at week 26 by αLOX-1.Envgp140 and αCD40.Env gp140 fusion protein administration IFNγ ELISPOTresponses summed over all antigens were by the Wilcoxon rank sum testshowed G1 vs. G3 (p=0.699), G2 vs. G4 (p=0.065), G1 vs. G2 (p=0.180), G3vs. G4 (p=0.818) and G4 vs. G5 (p=0.143). For the week 32 data theanalysis showed G1 vs. G3 (p=0.699), G2 vs. G4 (p=0.009*), G1 vs. G2(p=0.015*), G3 vs. G4 (p=1.000) and G4 vs. G5 (p=0.010*).

FIG. 7. Specificity and magnitude of blood HIV-1 antigen-specific CD4+ Tcell responses elicited by αLOX-1.Env gp140 and αCD40.Env gp140 fusionprotein administration. PBMCs from blood collected at week 26, and 32were analyzed by ICS for CD4+ T cell responses to HIV-1 antigen peptidepools (indicated above the graph). In contrast to FIG. 6, these datacombine IFNγ, IL-2, and TNFα cytokine secretion responses as well asintegrating responses across All Env (the response patterns representedwith combined cytokine responses were similar to those seen when theindividual cytokine responses were plotted). Dots are results forindividual animals and the box plots represent the distribution ofvalues with the bars indicating the median values for the responders inthe group (indicated below each panel).

FIG. 8. Specificity and magnitude of blood HIV-1 antigen-specific CD8+ Tcell responses elicited by αLOX-1.Env gp140 and αCD40.Env gp140 fusionprotein administration. PBMCs from blood collected at week 26 and 32were analyzed by ICS for CD8+ T cell responses to HIV-1 antigen peptidepools (indicated above the graph). In contrast to FIG. 6, the datacombine IFNγ, IL-2, and TNFα cytokine secretion responses as well asintegrating responses across All Env (the response patterns representedwith combined cytokine responses were similar to those seen when theindividual cytokine responses were plotted). Dots are results forindividual animals and the box plots represent the distribution ofvalues with the bars indicating the median values for the responders inthe group (indicated below each panel).

FIG. 9A-B. Durability of plasma IgG binding (A) and serum neutralizing(B) antibody responses evaluated as proportion of change per week.Proportion of change per week is calculated as [(Response at durabilitytime point—response at peak time point)/Response at peak timepoint]/number of weeks between peak and durability time points, and isonly calculated for animals that showed positive response for peakimmunity time point. The mid-line of the box denotes the median, and theends of the box denote the 25th and 75th percentiles. The whiskers thatextend from the top and bottom of the box extend to the most extremedata points that are no more than 1.5 times the interquartile range(i.e., the height of the box) or if no value meets this criterion, tothe data extremes. For binding antibody response, AUC from titrationdata in BAMA are used for the calculation. ID50 titers are used forneutralization response. Descriptive statistics of these values andresults for between-group comparisons are in Table 5.

FIG. 10A-D. Pair-wise comparison of magnitude of binding (A),neutralizing (B), ADCC (C), and T cell responses (D) between DC-targetedgp140 boost regimens (G1-G5 current study and ExtDC-N2Lp3) and thenon-DC-targeting gp120 boost regimen ExtNDC-N2[NP]2 at peak immunitytime point. In particular note, IgG binding to Con S gp140 CF for theExtNDC-N2[NP]2 group was 4 to 11-fold higher than G1-G5 in the currentstudy, with P values ≤0.0002 for each group in pair-wise comparison(2-tailed t test) (Table 6). IgG binding to gp70-B.CaseA V1V2 scaffoldfor the ExtNDC-N2[NP]2 animals was 4 to 27-fold that of G1-G5 in thecurrent study with P values <0.01 in pair-wise comparison (2-tailed ttest). IgA response was also significantly higher for the ExtNDC-N2[NP]2animals compared to G1-G5 in the current study (Table 6). For B valuesare significantly higher in the ExtNDC-N2[NP]2 group compared to G1-G5with P values <0.01 for most (2-tailed t test) (Table 7). The highestdifference was seen against Tier 1A clade C MW965.26, whereExtNDC-N2[NP]2 showed 7 to 29-fold higher ID50 titer compared to G1-G5of current study. ADCC response measured with Env coated cells wassignificantly higher for ExtNDC-N2[NP]2 compared to all 5 groups incurrent study at week 26. The differences ranged from 3-12 fold for AUCvalues (Table 8).

FIG. 11A-B. Durability of binding (A) and neutralizing (B) antibodyresponses evaluated as proportion of change per week. See FIG. 9 legendfor calculation of proportion of change per week. For binding antibodyresponse, AUC from titration data in BAMA are used for the calculation.ID50 titers are used for neutralization response. Boxes show 95%confidence interval and thick bars show mean of group. Proportion ofchange per week was only calculated for animals that showed positiveresponse for both peak and durability time points. Descriptivestatistics of these values and for values on additional antigens testedin BAMA are in Table 10.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As shown in the Examples of the application, anti-CD40-env gp140 fusionproteins increased B cell and T cell responses in vaccinated animals.The responses were robust, cross-reactive, and contained antibodiesspecific to multiple epitopes within gp140 including the C1, C2, V1-3,C4, C5, and gp41 immuno-dominant regions. The DC-targeting vaccines alsoelicited modest serum Env-specific IgA responses. Furthermore, CD4⁺ andCD8⁺ T cell responses specific to multiple Env epitopes were stronglyboosted by the DC-targeting vaccines administered in combination withpoly ICLC.

I. Nucleic Acids

In certain embodiments, there are recombinant nucleic acids encoding theproteins, polypeptides, or peptides described herein. Polynucleotidecontemplated for use in methods and compositions include those encodingantibodies to CD40 or binding portions thereof.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that either is recombinant or has been isolatedfree of total genomic nucleic acid. Included within the term“polynucleotide” are oligonucleotides (nucleic acids 100 residues orfewer in length), recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like. Polynucleotides include, incertain aspects, regulatory sequences, isolated substantially away fromtheir naturally occurring genes or protein encoding sequences.Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be RNA, DNA (genomic, cDNA or synthetic),analogs thereof, or a combination thereof. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide.

In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” isused to refer to a nucleic acid that encodes a protein, polypeptide, orpeptide (including any sequences required for proper transcription,post-translational modification, or localization). As will be understoodby those in the art, this term encompasses genomic sequences, expressioncassettes, cDNA sequences, and smaller engineered nucleic acid segmentsthat express, or may be adapted to express, proteins, polypeptides,domains, peptides, fusion proteins, and mutants. A nucleic acid encodingall or part of a polypeptide may contain a contiguous nucleic acidsequence encoding all or a portion of such a polypeptide. It also iscontemplated that a particular polypeptide may be encoded by nucleicacids containing variations having slightly different nucleic acidsequences but, nonetheless, encode the same or substantially similarprotein (see above).

In particular embodiments, there are isolated nucleic acid segments andrecombinant vectors incorporating nucleic acid sequences that encode apolypeptide (e.g., an antibody or fragment thereof) that binds to DCreceptors, such as CD40. The term “recombinant” may be used inconjunction with a polypeptide or the name of a specific polypeptide,and this generally refers to a polypeptide produced from a nucleic acidmolecule that has been manipulated in vitro or that is a replicationproduct of such a molecule.

The nucleic acid segments, regardless of the length of the codingsequence itself, may be combined with other nucleic acid sequences, suchas promoters, polyadenylation signals, additional restriction enzymesites, multiple cloning sites, other coding segments, and the like, suchthat their overall length may vary considerably. It is thereforecontemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant nucleic acid protocol.In some cases, a nucleic acid sequence may encode a polypeptide sequencewith additional heterologous coding sequences, for example to allow forpurification of the polypeptide, transport, secretion,post-translational modification, or for therapeutic benefits such astargeting or efficacy. As discussed above, a tag or other heterologouspolypeptide may be added to the modified polypeptide-encoding sequence,wherein “heterologous” refers to a polypeptide that is not the same asthe modified polypeptide.

In certain embodiments, there are polynucleotide variants havingsubstantial identity to the sequences disclosed herein; those comprisingat least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequenceidentity, including all values and ranges there between, compared to apolynucleotide sequence provided herein using the methods describedherein (e.g., BLAST analysis using standard parameters). In certainaspects, the isolated polynucleotide will comprise a nucleotide sequenceencoding a polypeptide that has at least 90%, preferably 95% and above,identity to an amino acid sequence described herein, over the entirelength of the sequence; or a nucleotide sequence complementary to saidisolated polynucleotide.

A. Vectors

Polypeptides may be encoded by a nucleic acid molecule. The nucleic acidmolecule can be in the form of a nucleic acid vector. The term “vector”is used to refer to a carrier nucleic acid molecule into which aheterologous nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated and expressed. A nucleic acid sequencecan be “heterologous,” which means that it is in a context foreign tothe cell in which the vector is being introduced or to the nucleic acidin which is incorporated, which includes a sequence homologous to asequence in the cell or nucleic acid but in a position within the hostcell or nucleic acid where it is ordinarily not found. Vectors includeDNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses,and plant viruses), and artificial chromosomes (e.g., YACs). One ofskill in the art would be well equipped to construct a vector throughstandard recombinant techniques (for example Sambrook et al., 2001;Ausubel et al., 1996, both incorporated herein by reference). Vectorsmay be used in a host cell to produce an antibody or fusion protein ofthe disclosure.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described herein.

B. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a recombinant protein-encoding sequence,is transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

In certain embodiments the host cell may be any prokaryotic oreukaryotic cell. In some aspects the host cell is an animal cell.Exemplary host cells include A549, B-cells, B16, BHK-21, C2C12, C6,CaCo-2, CAP/, CAP-T, CHO, CHO2, CHO-DG44, CHO-K1, COS-1, Cos-7, CV-1,Dendritic cells, DLD-1, Embryonic Stem (ES) Cell or derivative, H1299,HEK, 293, 293T, 293FT, Hep G2, Hematopoietic Stem Cells, HOS, Huh-7,Induced Pluripotent Stem (iPS) Cell or derivative, Jurkat, K562, L5278Y,LNCaP, MCF7, MDA-MB-231, MDCK, Mesenchymal Cells, Min-6, Monocytic cell,Neuro2a, NIH 3T3, NIH3T3L1, K562, NK-cells, NSO, Panc-1, PC12, PC-3,Peripheral blood cells, Plasma cells, Primary Fibroblasts, RBL, Renca,RLE, SF21, SF9, SH-SY5Y, SK-MES-1, SK—N—SH, SL3, SW403,Stimulus-triggered Acquisition of Pluripotency (STAP) cell or derivateSW403, T-cells, THP-1, Tumor cells, U2OS, U937, peripheral bloodlymphocytes, expanded T cells, hematopoietic stem cells, or Vero cells.

In some embodiments, cells may be subjected to limiting dilution methodsto enable the expansion of clonal populations of cells. The methods oflimiting dilution cloning are well known to those of skill in the art.Such methods have been described, for example for hybridomas but can beapplied to any cell. Such methods are described in (Cloning hybridomacells by limiting dilution, Journal of tissue culture methods, 1985,Volume 9, Issue 3, pp 175-177, by Joan C. Rener, Bruce L. Brown, andRoland M. Nardone) which is incorporated by reference herein.

In other embodiments, media may be formulated using componentswell-known to those skilled in the art. Formulations and methods ofculturing cells are described in detail in the following references:Short Protocols in Cell Biology J. Bonifacino, et al., ed., John Wiley &Sons, 2003, 826 pp; Live Cell Imaging: A Laboratory Manual D. Spector &R. Goldman, ed., Cold Spring Harbor Laboratory Press, 2004, 450 pp.;Stem Cells Handbook S. Sell, ed., Humana Press, 2003, 528 pp.; AnimalCell Culture: Essential Methods, John M. Davis, John Wiley & Sons, Mar.16, 2011; Basic Cell Culture Protocols, Cheryl D. Helgason, CindyMiller, Humana Press, 2005; Human Cell Culture Protocols, Series:Methods in Molecular Biology, Vol. 806, Mitry, Ragai R.; Hughes, RobinD. (Eds.), 3rd ed. 2012, XIV, 435 p. 89, Humana Press; Cancer CellCulture: Method and Protocols, Cheryl D. Helgason, Cindy Miller, HumanaPress, 2005; Human Cell Culture Protocols, Series: Methods in MolecularBiology, Vol. 806, Mitry, Ragai R.; Hughes, Robin D. (Eds.), 3rd ed.2012, XIV, 435 p. 89, Humana Press; Cancer Cell Culture: Method andProtocols, Simon P. Langdon, Springer, 2004; Molecular Cell Biology. 4thedition, Lodish H, Berk A, Zipursky S L, et al., New York: W. H.Freeman; 2000, Section 6.2 Growth of Animal Cells in Culture, all ofwhich are incorporated herein by reference.

C. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with an embodiment to produce nucleicacid sequences, or their cognate polypeptides, proteins and peptides.Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems, other examples ofexpression systems include STRATAGENE®'s COMPLETE CONTROL InducibleMammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

II. Proteinaceous Compositions

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table, below).

Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ sequences, respectively, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding region.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved, second orfurther-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. It is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillproduce a biologically equivalent and immunologically equivalentprotein.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine.

It is contemplated that in compositions there is between about 0.001 mgand about 10 mg of total polypeptide, peptide, and/or protein per ml.Thus, the concentration of protein in a composition can be about, atleast about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or anyrange derivable therein).

A. Polypeptides and Polypeptide Production

Embodiments involve polypeptides, peptides, and proteins and immunogenicfragments thereof for use in various aspects described herein. Forexample, specific antibodies are assayed for or used in binding to DCreceptors, such as CD40, and presenting HIV antigens. In specificembodiments, all or part of proteins described herein can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Alternatively, recombinant DNA technology may be employedwherein a nucleotide sequence that encodes a peptide or polypeptide isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

One embodiment includes the use of gene transfer to cells, includingmicroorganisms, for the production and/or presentation of proteins. Thegene for the protein of interest may be transferred into appropriatehost cells followed by culture of cells under the appropriateconditions. A nucleic acid encoding virtually any polypeptide may beemployed. The generation of recombinant expression vectors, and theelements included therein, are discussed herein. Alternatively, theprotein to be produced may be an endogenous protein normally synthesizedby the cell used for protein production.

In a certain aspects a DC receptor fragment comprises substantially allof the extracellular domain of a protein which has at least 85%identity, at least 90% identity, at least 95% identity, or at least97-99% identity, including all values and ranges there between, to asequence selected over the length of the fragment sequence.

Also included in immunogenic compositions are fusion proteins composedof HIV antigens, or immunogenic fragments of HIV antigens (e.g., gp140).HIV antigens may be from any type (e.g. HIV-1, HIV-2), group (e.g. groupM, group N, group O, or group P), sub-type or clade (e.g. clade A, B, C,D, F, G, H, J, K) or circulating recombinant form of HIV. Alternatively,embodiments also include individual fusion proteins of HIV proteins orimmunogenic fragments thereof, as a fusion protein with heterologoussequences such as a provider of T-cell epitopes or purification tags,for example: β-galactosidase, glutathione-S-transferase, greenfluorescent proteins (GFP), epitope tags such as FLAG, myc tag, polyhistidine, or viral surface proteins such as influenza virushaemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheriatoxoid, CRM197.

B. Antibodies and Antibody-Like Molecules

In certain aspects, one or more antibodies or antibody-like molecules(e.g., polypeptides comprising antibody CDR domains) may be obtained orproduced which have a specificity for Dendritic cell receptor, e.g.CD40, LOX-1, DCIR or langerin. These antibodies may be used in thetherapeutic applications described herein.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof, that can complete with the intact antibody forspecific binding to the target antigen, and includes chimeric,humanized, fully human, and bispecific antibodies. An intact antibodygenerally will comprise at least two full-length heavy chains and twofull-length light chains, but in some instances may include fewer chainssuch as antibodies naturally occurring in camelids which may compriseonly heavy chains. Antibodies according to the disclosure may be derivedsolely from a single source, or may be “chimeric,” that is, differentportions of the antibody may be derived from two different antibodies.For example, the CDR regions may be derived from a rat or murine source,while the framework region of the V region is derived from a differentanimal source, such as a human. The antibodies or binding fragments ofthe disclosure may be produced in hybridomas, by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Unless otherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable region domain(VL), and a constant region domain (CL). The variable region domain ofthe light chain is at the amino-terminus of the polypeptide. Lightchains according to the disclosure include kappa chains and lambdachains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable region domain(VH), and three constant region domains (CH1, CH2, and CH3). The VHdomain is at the amino-terminus of the polypeptide, and the CH domainsare at the carboxy-terminus, with the CH3 being closest to the —COOHend. Heavy chains according to the invention may be of any isotype,including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA(including IgA1 and IgA2 subtypes), IgM, and IgE.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” or “CDR”,interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system. An amino acidsequence which is substantially the same as a heavy or light chain CDRexhibits a considerable amount or extent of sequence identity whencompared to a reference sequence and contributes favorably to specificbinding of an antigen bound specifically by an antibody having thereference sequence. Such identity is definitively known or recognizableas representing the amino acid sequence of the particular humanmonoclonal antibody. Substantially the same heavy and light chain CDRamino acid sequence can have, for example, minor modifications orconservative substitutions of amino acids so long as the ability to binda particular antigen is maintained.

The term “CDR” or “complementarity determining region” means thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987) and additionally by MacCallumet al., J. Mol. Biol. 262:732-745 (1996), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orfunctional fragment thereof is intended to be within the scope of theterm as defined and used herein. The exact amino acid residue numberswhich encompass a particular CDR will vary depending on the structure ofthe CDR. Those skilled in the art can routinely determine which residuescomprise a particular CDR given the variable region amino acid sequenceof the antibody. Those skilled in the art can compare two or moreantibody sequences by defining regions or individual amino acidpositions of the respective sequences with the same CDR definition.

The term “antibody” includes both glycosylated and non-glycosylatedimmunoglobulins of any isotype or subclass or combination thereof,including human (including CDR-grafted antibodies), humanized, chimeric,multi-specific, monoclonal, polyclonal, and oligomers thereof,irrespective of whether such antibodies are produced, in whole or inpart, via immunization, through recombinant technology, by way of invitro synthetic means, or otherwise. Thus, the term “antibody” includesthose that are prepared, expressed, created or isolated by recombinantmeans, such as (a) antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes or a hybridomaprepared therefrom, (b) antibodies isolated from a host cell transfectedto express the antibody, (c) antibodies isolated from a recombinant,combinatorial library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of immunoglobulingene sequences of two distinct species of animals. In certainembodiments, however, such antibodies can be subjected to in vitromutagenesis (or, when an animal transgenic for human immunoglobulinsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the antibodies are sequences that,while derived from and related to the germline VH and VL sequences of aparticular species (e.g., human), may not naturally exist within thatspecies' antibody germline repertoire in vivo.

The term “antigen-binding fragment” of an antibody means one or morefragments of an antibody that retain the ability to specifically bind toan antigen that is specifically bound by a reference antibody, asdisclosed herein. An “antigen-binding fragment” of an antibody mayinclude, for example, polypeptides comprising individual heavy or lightchains and fragments thereof, such as VL, VH, and Fd regions (consistingof the VH and CH1 domains); monovalent fragments, such as Fv, Fab, andFab′ regions; bivalent fragments, such as F(ab′)2; single chainantibodies, such as single chain Fv (scFv) regions; Fc fragments;diabodies; maxibodies (bivalent scFv fused to the amino terminus of theFc (CH2-CH3 domains)) and complementary determining region (CDR)domains. Such terms are described, for example, in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N Y(1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference(Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., CellBiophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol.,178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, 2d ed.,Wiley-Liss, Inc. New York, N.Y. (1990), which are incorporated herein byreference.

The term “antigen-binding fragment” also includes, for example,fragments produced by protease digestion or reduction of a humanmonoclonal antibody and by recombinant DNA methods known to thoseskilled in the art. One skilled in the art knows that the exactboundaries of a fragment of a human monoclonal antibody can be variable,so long as the fragment maintains a functional activity. Usingwell-known recombinant methods, one skilled in the art can engineer anucleic acid to express a functional fragment with any endpoints desiredfor a particular application. Furthermore, although the two domains ofthe Fv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingfragment” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies. Such fragments include those obtained by amino-terminaland/or carboxy-terminal deletions, but where the remaining amino acidsequence is substantially identical to the corresponding positions inthe naturally-occurring sequence deduced, for example, from afull-length cDNA sequence. Antigen-binding fragments also includefragments of an antibody which retain at least one (e.g., 1, 2, 3 ormore) light chain sequences for a particular complementarity determiningregion (CDR) (e.g., at least one or more of CDR1, CDR2, and/or CDR3 fromthe heavy and/or light chain). Fusions of CDR containing sequences to anFc region (or a CH2 or CH3 region thereof) are included within the scopeof this definition including, for example, scFv fused, directly orindirectly, to an Fc region are included herein. An antigen-bindingfragment is inclusive of, but not limited to, those derived from anantibody or fragment thereof (e.g., by enzymatic digestion or reductionof disulfide bonds), produced synthetically using recombinant methods,created via in vitro synthetic means (e.g., Merrifield resins),combinations thereof, or through other methods. Antigen-bindingfragments may also comprise multiple fragments, such as CDR fragments,linked together synthetically, chemically, or otherwise, in the form ofoligomers. Thus, antigen-binding fragments of the present inventioninclude polypeptides produced by any number of methods which comprise atleast one CDR from a VH or VL chain of the present invention (e.g.,derived from monoclonal antibodies 480.12 and 994.1).

The term “VL fragment” means a fragment of the light chain of amonoclonal antibody which includes all or part of the light chainvariable region, including the CDRs. A VL fragment can further includelight chain constant region sequences.

The term “VH fragment” means a fragment of the heavy chain of amonoclonal antibody which includes all or part of the heavy chainvariable region, including the CDRs. A VH fragment can further includeheavy chain constant region sequences.

The term “Fd fragment” means a fragment of the heavy chain of amonoclonal antibody which includes all or part of the VH heavy chainvariable region, including the CDRs. An Fd fragment can further includeCH1 heavy chain constant region sequences.

An “Fc” region contains two heavy chain fragments comprising the CH1 andCH2 domains of an antibody. The two heavy chain fragments are heldtogether by two or more disulfide bonds and by hydrophobic interactionsof the CH3 domain.

The term “Fv fragment” means a monovalent antigen-binding fragment of amonoclonal antibody, including all or part of the variable regions ofthe heavy and light chains, and absent of the constant regions of theheavy and light chains. The variable regions of the heavy and lightchains include, for example, the CDRs.

The term “Fab fragment” means a monovalent antigen-binding fragment ofan antibody consisting of the VL, VH, CL and CH1 domains, which islarger than an Fv fragment. For example, a Fab fragment includes thevariable regions, and all or part of the first constant domain of theheavy and light chains.

The term “Fab′ fragment” means a monovalent antigen-binding fragment ofa monoclonal antibody that is larger than a Fab fragment. For example, aFab′ fragment includes all of the light chain, all of the variableregion of the heavy chain, and all or part of the first and secondconstant domains of the heavy chain.

The term “F(ab′)2 fragment” means a bivalent antigen-binding fragment ofa monoclonal antibody comprising two Fab fragments linked by a disulfidebridge at the hinge region. An F(ab′)2 fragment includes, for example,all or part of the variable regions of two heavy chains and two lightchains, and can further include all or part of the first constantdomains of two heavy chains and two light chains.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding fragment.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are herein incorporated byreference.

A “domain antibody” is an antigen-binding fragment containing only thevariable region of a heavy chain or the variable region of a lightchain. In some instances, two or more VH regions are covalently joinedwith a peptide linker to create a bivalent domain antibody. The two VHregions of a bivalent domain antibody may target the same or differentantigens.

The term “bivalent antibody” means an antibody that comprises twoantigen binding sites. In some instances, the two binding sites have thesame antigen specificities. However, bivalent antibodies may bebispecific.

The term “bispecific antibody” means an antibody that binds to two ormore distinct epitopes. For example, the antibody may bind to, orinteract with, (a) a cell surface antigen and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific antibody” or“heterospecific antibody” means an antibody that binds to more than twodistinct epitopes. For example, the antibody may bind to, or interactwith, (a) a cell surface antigen, (b) an Fc receptor on the surface ofan effector cell, and (c) at least one other component.

Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific antibodies orantigen-binding fragments thereof which are directed to certain epitopesand to other targets, such as Fc receptors on effector cells. Bispecificantibodies are a species of multispecific antibody and may be producedby a variety of methods including, but not limited to, fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai andLachmann, Clin. Exp. Immunol. 79:315 (1990); Kostelny et al., J.Immunol. 148:1547 (1992). The two binding sites of a bispecific antibodywill bind to two different epitopes, which may reside on the same ordifferent protein targets. The term “bispecific antibodies” alsoincludes diabodies. Diabodies are bivalent, bispecific antibodies inwhich the VH and VL domains are expressed on a single polypeptide chain,but using a linker that is too short to allow for pairing between thetwo domains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Hollinger et al., Proc Natl. Acad. Sci. USA90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994).

The term “monoclonal antibody” or “mAb,” as used herein, refers to anantibody obtained from a population of substantially homogeneousantibodies, e.g., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. In contrast to polyclonal antibodypreparations that typically include different antibodies againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. The term is not limitedregarding the species or source of the antibody, nor is it intended tobe limited by the manner in which it is made. The term encompasses wholeimmunoglobulins as well as fragments such as Fab, F(ab′)2, Fv, and otherfragments, as well as chimeric and humanized homogeneous antibodypopulations, that exhibit immunological binding properties of the parentmonoclonal antibody molecule.

The term “mouse monoclonal antibody” means a monoclonal antibody, asdefined above, produced by immunizing a mouse, with an antigen ofinterest. A “mouse monoclonal antibody” is produced using conventionalmethods well known in the art, from mouse-mouse hybridomas, describedmore fully below.

The term “rabbit monoclonal antibody” as used herein means a monoclonalantibody, as defined above, produced by immunizing a rabbit with anantigen of interest. A “rabbit monoclonal antibody” can be producedusing rabbit-rabbit hybridomas (e.g., fusions between anantibody-producing cell from the immunized rabbit with an immortalizedcell from a rabbit), rabbit-mouse hybridomas (e.g., fusions between anantibody-producing cell from the immunized rabbit with an immortalizedcell from a mouse), and the like.

The term “human monoclonal antibody” means a monoclonal antibody withsubstantially human CDR amino acid sequences produced, for example, byrecombinant methods, by lymphocytes or by hybridoma cells.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851 (1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. See,e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswaniand Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris,Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op.Biotech. 5:428 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingregions.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al., Bio/Technology 10:779 (1992) describes affinity maturationby VH and VL domain shuffling. Random mutagenesis of CDR and/orframework residues is described by: Barbas et al., Proc. Natl. Acad.Sci. USA 91:3809 (1994); Schier et al., Gene 169:147 (1995); Yelton etal., J. Immunol. 155:1994 (1995); Jackson et al., J. Immunol. 154:3310(1995); and Hawkins et al., J. Mol. Biol. 226:889 (1992).

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to that antigen. An antigen may possessone or more epitopes that are capable of interacting with differentantibodies.

The term “epitope” includes any determinant capable of binding with highaffinity to an immunoglobulin or to a T-cell receptor. An epitope is aregion of an antigen that is bound by an antibody that specificallytargets that antigen, and when the antigen is a protein, includesspecific amino acids that directly contact the antibody. Most often,epitopes reside on proteins, but in some instances, may reside on otherkinds of molecules, such as nucleic acids. Epitope determinants mayinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and may havespecific three dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially recognize an epitope on the targetantigen in a complex mixture of proteins and/or macromolecules.

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182(1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography andtwo-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci. USA 78:3824-3828(1981) for determining antigenicity profiles, and the Kyte-Doolittletechnique, Kyte et al., J. Mol. Biol. 157:105-132 (1982) for hydropathyplots.

An antibody of the disclosure is said to “specifically bind” its targetantigen when the dissociation constant (K_(D)) is ≤10⁻⁸ M. The antibodyspecifically binds antigen with “high affinity” when the K_(D) is≤5×10⁻⁹ M, and with “very high affinity” when the K_(D) is ≤5×10⁻¹° M.In one embodiment of the invention, the antibody has a K_(D) of ≤10⁻⁹ Mand an off-rate (K_(D)) of about 1×10⁻⁴/sec. In one embodiment of theinvention, the off-rate is ≤1×10⁻⁵/sec.

It is understood that the antibodies of the present disclosure may bemodified, such that they are substantially identical to the antibodypolypeptide sequences, or fragments thereof, and still bind the epitopesof the disclosure. Polypeptide sequences are “substantially identical”when optimally aligned using such programs as GAP or BESTFIT usingdefault gap weights, they share at least 80% sequence identity, at least90% sequence identity, at least 95% sequence identity, at least 96%sequence identity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity.

As discussed herein, minor variations in the amino acid sequences ofantibodies or antigen-binding regions thereof are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% and most preferably at least 99% sequence identity. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic (aspartate, glutamate);(2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan);and (4) uncharged polar (glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine). More preferred families are: (1)aliphatic-hydroxy (serine, threonine); (2) amide-containing (asparagine,glutamine); (3) aliphatic (alanine, valine, leucine, isoleucine); and(4) aromatic (phenylalanine, tryptophan). For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal., Science 253:164 (1991).

The antibodies of the present disclosure may also be generated usingpeptide analogs of the epitopic determinants disclosed herein, whichanalogs may consist of non-peptide compounds having properties analogousto those of the template peptide. These types of non-peptide compoundare termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv.Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); andEvans et al., J. Med. Chem. 30:1229 (1987).

“Mini-antibodies” or “minibodies” are also contemplated for use withembodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region (Pack, et al., 1992). The oligomerization domain comprisesself-associating α-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al., 2003 describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Alternative scaffolds for antigen binding peptides, such as CDRs arealso available and can be used to generate DC receptor-binding moleculesin accordance with the embodiments. Generally, a person skilled in theart knows how to determine the type of protein scaffold on which tograft at least one of the CDRs arising from the original antibody. Moreparticularly, it is known that to be selected such scaffolds must meetthe greatest number of criteria as follows (Skerra, 2000): goodphylogenetic conservation; known three-dimensional structure (as, forexample, by crystallography, NMR spectroscopy or any other techniqueknown to a person skilled in the art); small size; few or nopost-transcriptional modifications; and/or easy to produce, express andpurify.

The origin of such protein scaffolds can be, but is not limited to, thestructures selected among: fibronectin and preferentially fibronectintype III domain 10, lipocalin, anticalin (Skerra, 2001), protein Zarising from domain B of protein A of Staphylococcus aureus, thioredoxinA or proteins with a repeated motif such as the “ankyrin repeat” (Kohlet al., 2003), the “armadillo repeat”, the “leucine-rich repeat” and the“tetratricopeptide repeat”. For example, anticalins or lipocalinderivatives are a type of binding proteins that have affinities andspecificities for various target molecules and can be used as DCreceptor-binding molecules. Such proteins are described in US PatentPublication Nos. 20100285564, 20060058510, 20060088908, 20050106660, andPCT Publication No. WO2006/056464, incorporated herein by reference.

Scaffolds derived from toxins such as, for example, toxins fromscorpions, insects, plants, mollusks, etc., and the protein inhibitersof neuronal NO synthase (PIN) may also be used in certain aspects.

Chimeric and humanized antibodies based upon the foregoing sequences arealso encompassed by the current disclosure. Monoclonal antibodies foruse as therapeutic agents may be modified in various ways prior to use.One example is a “chimeric” antibody, which is an antibody composed ofprotein segments from different antibodies that are covalently joined toproduce functional immunoglobulin light or heavy chains orantigen-binding fragments thereof. Generally, a portion of the heavychain and/or light chain is identical with, or homologous to, acorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is/are identical or homologous to acorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. For methods relating tochimeric antibodies, see, for example, U.S. Pat. No. 4,816,567; andMorrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), whichare hereby incorporated by reference. CDR grafting is described, forexample, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089,and 5,530,101, which are all hereby incorporated by reference for allpurposes.

Generally, the goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended patent species ismaximized. One example is the “CDR-grafted” antibody, in which theantibody comprises one or more complementarity determining regions(CDRs) from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, the V region or selected CDRs from a rodentantibody often are grafted into a human antibody, replacing thenaturally-occurring V regions or CDRs of the human antibody.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody or corresponding isotype. Preferably,humanized antibodies contain minimal sequence derived from non-humanimmunoglobulin sequences. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205. In some instances, frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762). Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance (e.g., to obtain desired affinity). In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details see Jones et al., Nature 331:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992); Verhoeyen et al., Science 239:1534-36 (1988)).

In one aspect of the disclosure, the CDRs of the light and heavy chainvariable regions of the antibodies provided herein are grafted toframework regions (FRs) from antibodies from the same, or a different,phylogenetic species. To create consensus human FRs, FRs from severalhuman heavy chain or light chain amino acid sequences may be aligned toidentify a consensus amino acid sequence. In one aspect of theinvention, rare amino acids in the FRs of the heavy and light chains ofthe antibody are not replaced, while the rest of the FR amino acids arereplaced. A “rare amino acid” is a specific amino acid that is in aposition in which this particular amino acid is not usually found in anFR. Alternatively, the grafted variable regions from the antibody may beused with a constant region that is different from the constant regionof antibody. In other embodiments of the disclosure, the graftedvariable regions are part of a single chain Fv antibody.

In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

Also encompassed are xenogeneic or modified antibodies produced in anon-human mammalian host, more particularly a transgenic mouse,characterized by inactivated endogenous immunoglobulin (Ig) loci. Insuch transgenic animals, competent endogenous genes for the expressionof light and heavy subunits of host immunoglobulins are renderednon-functional and substituted with the analogous human immunoglobulinloci. These transgenic animals produce human antibodies in thesubstantial absence of light or heavy host immunoglobulin subunits. See,for example, U.S. Pat. No. 5,939,598.

Fully human antibodies are also provided. Methods are available formaking fully human antibodies specific for a given antigen withoutexposing human beings to the antigen (“fully human antibodies”). Onemeans for implementing the production of fully human antibodies is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated is one means of producing fully human monoclonalantibodies (mAbs) in mouse, an animal that can be immunized with anydesirable antigen. Using fully human antibodies can minimize theimmunogenic and allergic responses that can sometimes be caused byadministering mouse or mouse-derivatized mAbs to humans as therapeuticagents.

In one embodiment, human antibodies may be produced in a non-humantransgenic animal, e.g., a transgenic mouse capable of producingmultiple isotypes of human antibodies to CD40 (e.g., IgG, IgA, and/orIgE) by undergoing V-D-J recombination and isotype switching.Accordingly, aspects of the disclosure include not only antibodies,antibody fragments, and pharmaceutical compositions thereof, but alsonon-human transgenic animals, B-cells, host cells, and hybridomas whichproduce the antibodies. The present disclosure further encompassespharmaceutical preparations containing the antibodies, and methods oftreating physiological disorders, e.g., HIV, by administering theantibodies of the present disclosure.

Fully human antibodies can be produced by immunizing transgenic animals(usually mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA90:2551-2555 (1993); Jakobovits et al., Nature 362:255-258 (1993);Bruggermann et al., Year in Immunol. 7:33 (1993). In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, for example,International Patent Application Publication Nos. WO 96/33735 and WO94/02602, which are hereby incorporated by reference in their entirety.Additional methods relating to transgenic mice for making humanantibodies are described in U.S. Pat. Nos. 5,545,807; 6,713,610;6,673,986; 6,162,963; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and5,545,806; in International Patent Application Publication Nos. WO91/10741 and WO 90/04036; and in European Patent Nos. EP 546073B1 and EP546073A1, all of which are hereby incorporated by reference in theirentirety for all purposes.

The transgenic mice described above, referred to herein as “HuMAb” mice,contain a human immunoglobulin gene minilocus that encodes unrearrangedhuman heavy (μ and γ) and κ light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg et al., Nature 368:856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or κ chains and in responseto immunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgG kappa monoclonal antibodies (Lonberg et al., supra; Lonbergand Huszar, Intern. Ref. Immunol. 13:65-93 (1995); Harding and Lonberg,Ann. N.Y. Acad. Sci. 764:536-546 (1995)). The preparation of HuMAb miceis described in detail in Taylor et al., Nucl. Acids Res. 20:6287-6295(1992); Chen et al., Int. Immunol. 5:647-656 (1993); Tuaillon et al., J.Immunol. 152:2912-2920 (1994); Lonberg et al., supra; Lonberg, Handbookof Exp. Pharmacol. 113:49-101 (1994); Taylor et al., Int. Immunol.6:579-591 (1994); Lonberg and Huszar, Intern. Ref. Immunol. 13:65-93(1995); Harding and Lonberg, Ann. N.Y. Acad. Sci. 764:536-546 (1995);Fishwild et al., Nat. Biotechnol. 14:845-851 (1996); the foregoingreferences are herein incorporated by reference in their entirety forall purposes. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; 5,770,429; and 5,545,807; as well as International PatentApplication Publication Nos. WO 93/1227; WO 92/22646; and WO 92/03918,the disclosures of all of which are hereby incorporated by reference intheir entirety for all purposes. Technologies utilized for producinghuman antibodies in these transgenic mice are disclosed also in WO98/24893, and Mendez et al., Nat. Genetics 15:146-156 (1997), which areherein incorporated by reference.

Using hybridoma technology, antigen-specific human mAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies may be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(as disclosed in Hoogenboom et al., J. Mol. Biol. 227:381 (1991); andMarks et al., J. Mol. Biol. 222:581 (1991)). Phage-display techniquesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in International Patent Application Publication No. WO99/10494 (herein incorporated by reference), which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three-dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for CD40 binding, thus yielding informationgathered from such routine experiments, one skilled in the art canreadily determine the amino acid positions where further substitutionsshould be avoided either alone or in combination with other mutations.

In some embodiments of the disclosure, amino acid substitutions are madethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter ligand or antigen binding affinities,and/or (5) confer or modify other physicochemical or functionalproperties on such polypeptides. For example, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally-occurring sequence.Substitutions can be made in that portion of the antibody that liesoutside the domain(s) forming intermolecular contacts. In suchembodiments, conservative amino acid substitutions can be used that donot substantially change the structural characteristics of the parentsequence (e.g., one or more replacement amino acids that do not disruptthe secondary structure that characterizes the parent or nativeantibody). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples (Creighton, Ed.), 1984, W.H. New York: Freeman and Company;Introduction to Protein Structure (Brandon and Tooze, eds.), 1991 NewYork: Garland Publishing; and Thornton et al., Nature 354:105 (1991),each of which is incorporated herein by reference in its entirety forall purposes.

The disclosure also encompasses glycosylation variants of the antibodieswherein the number and/or type of glycosylation site(s) has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, antibody protein variants comprise a greater or alesser number of N-linked glycosylation sites than the native antibody.An N-linked glycosylation site is characterized by the sequence:Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as Xmay be any amino acid residue except proline. The substitution of aminoacid residues to create this sequence provides a potential new site forthe addition of an N-linked carbohydrate chain. Alternatively,substitutions that eliminate or alter this sequence will preventaddition of an N-linked carbohydrate chain present in the nativepolypeptide. For example, the glycosylation can be reduced by thedeletion of an Asn or by substituting the Asn with a different aminoacid. In other embodiments, one or more new N-linked glycosylation sitesare created. Antibodies typically have a N-linked glycosylation site inthe Fc region.

Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues in the parent or native amino acidsequence are deleted from or substituted with another amino acid (e.g.,serine). Cysteine variants are useful, inter alia, when antibodies mustbe refolded into a biologically active conformation. Cysteine variantsmay have fewer cysteine residues than the native antibody, and typicallyhave an even number to minimize interactions resulting from unpairedcysteines.

Derivatives of the antibodies and antigen binding fragments that aredescribed herein are also provided. The derivatized antibody or fragmentmay comprise any molecule or substance that imparts a desired propertyto the antibody or fragment, such as increased half-life in a particularuse. The derivatized antibody can comprise, for example, a detectable(or labeling) moiety (e.g., a radioactive, colorimetric, antigenic orenzymatic molecule, a detectable bead [such as a magnetic orelectrodense (e.g., gold) bead], or a molecule that binds to anothermolecule (e.g., biotin or streptavidin), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antibodyfor a particular use (e.g., administration to a subject, such as a humansubject, or other in vivo or in vitro uses). Examples of molecules thatcan be used to derivatize an antibody include albumin (e.g., human serumalbumin) and polyethylene glycol (PEG). Albumin-linked and PEGylatedderivatives of antibodies can be prepared using techniques well known inthe art.

In certain embodiments, a polypeptide that specifically binds to DCreceptors is able to bind a DC receptor on the surface of the cells andpresent an HIV antigen that allows the generation of a robust immuneresponse. Moreover, in some embodiments, the polypeptide that is usedcan provided protective immunity against HIV.

1. Methods for Generating Antibodies

Methods for generating antibodies (e.g., monoclonal antibodies and/ormonoclonal antibodies) are known in the art. Briefly, a polyclonalantibody is prepared by immunizing an animal with an antigenicpolypeptide, such as a CD40 polypeptide or a portion thereof inaccordance with embodiments and collecting antisera from that immunizedanimal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art. Itwill be appreciated that antibodies can also be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include any acceptable immunostimulatory compound, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or vectors encoding such adjuvants.

Adjuvants that may be used in accordance with embodiments include, butare not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP,BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP,CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion is also contemplated. MHC antigens may even be used. Exemplaryadjuvants may include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxideadjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as -interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of antibodiesvaries upon the nature of the immunogen as well as the animal used forimmunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of antibodies may be monitored by sampling blood of theimmunized animal at various points following immunization.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, Rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages (Goding, 1986, pp. 60 61). Mice(e.g., BALB/c mice) are routinely used and generally give a highpercentage of stable fusions.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiescan be obtained from the monoclonal antibodies so produced by methodswhich include digestion with enzymes, such as pepsin or papain, and/orby cleavage of disulfide bonds by chemical reduction. Alternatively,monoclonal antibody fragments can be synthesized using an automatedpeptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 104 times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies.

Another embodiment concerns producing antibodies, for example, as isfound in U.S. Pat. No. 6,091,001, which describes methods to produce acell expressing an antibody from a genomic sequence of the cellcomprising a modified immunoglobulin locus using Cre-mediatedsite-specific recombination is disclosed. The method involves firsttransfecting an antibody-producing cell with a homology-targeting vectorcomprising a lox site and a targeting sequence homologous to a first DNAsequence adjacent to the region of the immunoglobulin loci of thegenomic sequence which is to be converted to a modified region, so thefirst lox site is inserted into the genomic sequence via site-specifichomologous recombination. Then the cell is transfected with alox-targeting vector comprising a second lox site suitable forCre-mediated recombination with the integrated lox site and a modifyingsequence to convert the region of the immunoglobulin loci to themodified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments can be synthesized using anautomated peptide synthesizer, or by expression of full-length gene orof gene fragments in E. coli.

It is further contemplated that monoclonal antibodies may be furtherscreened or optimized for properties relating to specificity, avidity,half-life, immunogenicity, binding association, binding disassociation,or overall functional properties relative to being a treatment forinfection. Thus, it is contemplated that monoclonal antibodies may have1, 2, 3, 4, 5, 6, or more alterations in the amino acid sequence of 1,2, 3, 4, 5, or 6 CDRs of monoclonal antibodies or humanized antibodiesprovided herein. It is contemplated that the amino acid in position 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3, CDR4, CDR5, or CDR6of the VJ or VDJ region of the light or heavy variable region ofantibodies may have an insertion, deletion, or substitution with aconserved or non-conserved amino acid. Such amino acids that can eitherbe substituted or constitute the substitution are disclosed above.

In some embodiments, fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment constituted with the VL, VH, CL and CH1 domains; (ii) theFd fragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconstituted with the VL and VH domains of a single antibody; (iv) thedAb fragment (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003),which is constituted with a VH or a VL domain; (v) isolated CDR regions;(vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988;Huston et al., 1988); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; Holliger et al.,1993). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996). Minibodies comprising a scFv joined to a CH3domain may also be made (Hu et al. 1996). The citations in thisparagraph are all incorporated by reference.

Antibodies also include bispecific antibodies. Bispecific orbifunctional antibodies form a second generation of monoclonalantibodies in which two different variable regions are combined in thesame molecule (Holliger & Winter, 1999). Their use has been demonstratedboth in the diagnostic field and in the therapy field from theircapacity to recruit new effector functions or to target severalmolecules on the surface of tumor cells. Where bispecific antibodies areto be used, these may be conventional bispecific antibodies, which canbe manufactured in a variety of ways (Holliger et al, 1993), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. These antibodies can beobtained by chemical methods (Glennie et al., 1987; Repp et al., 1995)or somatic methods (Staerz & Bevan, 1986) but likewise by geneticengineering techniques which allow the heterodimerization to be forcedand thus facilitate the process of purification of the antibody sought(Merchand et al., 1998). Examples of bispecific antibodies include thoseof the BiTE™ technology in which the binding domains of two antibodieswith different specificity can be used and directly linked via shortflexible peptides. This combines two antibodies on a short singlepolypeptide chain. Diabodies and scFv can be constructed without an Fcregion, using only variable domains, potentially reducing the effects ofanti-idiotypic reaction. The citations in this paragraph are allincorporated by reference.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against SpA, then a library can be made where the other arm isvaried and an antibody of appropriate specificity selected. Bispecificwhole antibodies may be made by alternative engineering methods asdescribed in Ridgeway et al., 1996), which is hereby incorporated byreference.

C. Antibody and Polypeptide Conjugates

Embodiments provide antibodies and antibody-like molecules against DCreceptor, e.g. CD40, polypeptides and peptides that are linked to atleast one agent to form an antibody conjugate or payload. In order toincrease the efficacy of antibody molecules as diagnostic or therapeuticagents, it is conventional to link or covalently bind or complex atleast one desired molecule or moiety. Such a molecule or moiety may be,but is not limited to, at least one effector or reporter molecule.Effector molecules comprise molecules having a desired activity, e.g.,cytotoxic activity. Non-limiting examples of effector molecules whichhave been attached to antibodies include toxins, therapeutic enzymes,antibiotics, radio-labeled nucleotides and the like. By contrast, areporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies include enzymes, radiolabels, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired.

Antibody conjugates include those intended primarily for use in vitro,where the antibody is linked to a secondary binding ligand and/or to anenzyme (an enzyme tag) that will generate a colored product upon contactwith a chromogenic substrate. Examples of suitable enzymes include, butare not limited to, urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Preferred secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In some embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In some embodiments anti-CD40 antibodies are used to target HIV antigensto dendritic cells. Compositions and methods for the expression,secretion and use of anti-CD40 antibodies as vaccines and antigendelivery vectors with one linked antigenic peptides are described in WO2010/104761; all methods disclosed are incorporated herein by reference.

In certain aspects, peptide linkers are used to link a dendritic cellspecific antibodies and HIV antigens to be presented. Peptide linkersmay incorporate glycosylation sites or introduce secondary structure.Additionally these linkers increase the efficiency of expression orstability of the fusion protein and as a result the efficiency ofantigen presentation to a dendritic cell. Such linkers may include theflexV1, f1, f2, and/or f3 linkers. These examples and others arediscussed in WO 2010/104747, the contents of which are incorporatedherein by reference.

D. Immunostimulants

In some embodiments an immunostimulant is administered in combinationwith the antibody-antigen fusion protein. The term “immunostimulant” asused herein refers to a compound that can stimulate an immune responsein a subject, and may include an adjuvant. In some embodiments, theimmunostimulant is directly fused to the CD40 specific antibody. Ineither case, the immunostimulant may enhance the efficacy of thevaccine. In certain aspects the immunostimulant may be a toll-likereceptor (TLR) agonist. TLR agonists comprise flagellins from Salmonellaenterica or Vibrio cholerae. TLR agonists may be specific for certainTLR classes (i.e., TLR5, TLR7 or TLR9 agonists) and may be presented inany combination or as any modification. Examples of such immuneadjuvants are described in WO 2012/021834, the contents of which areincorporated herein by reference.

In some embodiments, an immunostimulant is an agent that does notconstitute a specific antigen, but can boost the strength and longevityof an immune response to an antigen. Such immunostimulants may include,but are not limited to stimulators of pattern recognition receptors,such as Toll-like receptors, RIG-1 and NOD-like receptors (NLR), mineralsalts, such as alum, alum combined with monphosphoryl lipid (MPL) A ofEnterobacteria, such as Escherihia coli, Salmonella minnesota,Salmonella typhimurium, or Shigella flexneri or specifically with MPL®(AS04), MPL A of above-mentioned bacteria separately, saponins, such asQS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide,ISA 51 and ISA 720, AS02 (QS21+squalene+MPL.), liposomes and liposomalformulations such as ASO1, synthesized or specifically preparedmicroparticles and microcarriers such as bacteria-derived outer membranevesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, orchitosan particles, depot-forming agents, such as Pluronic blockco-polymers, specifically modified or prepared peptides, such as muramyldipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, orproteins, such as bacterial toxoids or toxin fragments.

In some embodiments, the immunostimulant comprises an agonist forpattern recognition receptors (PRR), including, but not limited toToll-Like Receptors (TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/orcombinations thereof. In some embodiments, additional agents compriseagonists for Toll-Like Receptors 3, agonists for Toll-Like Receptors 7and 8, or agonists for Toll-Like Receptor 9. In some embodiments, theimmunostimulants comprise imidazoquinolines; such as R848; adeninederivatives, such as those disclosed in U.S. Pat. No. 6,329,381, U.S.Published Patent Application 2010/0075995, or WO 2010/018132;immunostimulatory DNA; or immunostimulatory RNA. In some embodiments,the immunostimulant may comprise immunostimulatory RNA molecules, suchas but not limited to dsRNA, poly I:C, poly I:poly C, 12U (available asAmpligen®, both poly I:C and poly I:polyC 12U being known as TLR3stimulants), polyICLC (such as Hiltonol), and/or those disclosed in F.Heil et al., “Species-Specific Recognition of Single-Stranded RNA viaToll-like Receptor 7 and 8” Science 303(5663), 1526-1529 (2004); J.Vollmer et al., “Immune modulation by chemically modifiedribonucleosides and oligoribonucleotides” WO 2008033432 A2; A. Forsbachet al., “Immunostimulatory oligoribonucleotides containing specificsequence motif(s) and targeting the Toll-like receptor 8 pathway” WO2007062107 A2; E. Uhlmann et al., “Modified oligoribonucleotide analogswith enhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US2006241076; G. Lipford et al., “Immunostimulatory viral RNAoligonucleotides and use for treating cancer and infections” WO2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containingoligoribonucleotides, compositions, and screening methods” WO 2003086280A2. In some embodiments, an additional agent may be a TLR-4 agonist,such as bacterial lipopolysaccharide (LPS), VSV-G, and/or HMGB-1. Insome embodiments, additional agents may comprise TLR-5 agonists, such asflagellin, or portions or derivatives thereof, including but not limitedto those disclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and7,192,725.

In some embodiments, the immunostimulant may be proinflammatory stimulireleased from necrotic cells (e.g., urate crystals). In someembodiments, the immunostimulant may be activated components of thecomplement cascade (e.g., CD21, CD35, etc.). In some embodiments, theimmunostimulant may be activated components of immune complexes.Immunostimulants also include complement receptor agonists, such as amolecule that binds to CD21 or CD35. In some embodiments, the complementreceptor agonist induces endogenous complement opsonization of thesynthetic nanocarrier. In some embodiments, immunostimulants arecytokines, which are small proteins or biological factors (in the rangeof 5 kD-20 kD) that are released by cells and have specific effects oncell-cell interaction, communication and behavior of other cells. Insome embodiments, the cytokine receptor agonist is a small molecule,antibody, fusion protein, or aptamer.

III. Methods of Treatment

As discussed above, the compositions and methods of using thesecompositions can treat a subject (e.g., prevent an HIV infection orevoke a robust immune response to HIV) having, suspected of having, orat risk of developing an infection or related disease, particularlythose related to HIV.

As used herein the phrase “immune response” or its equivalent“immunological response” refers to a humoral (antibody mediated),cellular (mediated by antigen-specific T cells or their secretionproducts) or both humoral and cellular response directed against aprotein, peptide, or polypeptide of the invention in a recipientpatient. Treatment or therapy can be an active immune response inducedby administration of immunogen or a passive therapy effected byadministration of antibody, antibody containing material, or primedT-cells.

For purposes of this specification and the accompanying claims the terms“epitope,” “antigen,” “Ag,” and “antigenic determinant” are usedinterchangeably to refer to a site on an antigen to which B and/or Tcells respond or recognize. B-cell epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude those methods described in Epitope Mapping Protocols (1996). Tcells recognize continuous epitopes of about nine amino acids for CD8cells or about 13-15 amino acids for CD4 cells. T cells that recognizethe epitope can be identified by in vitro assays that measureantigen-dependent proliferation, as determined by 3H-thymidineincorporation by primed T cells in response to an epitope (Burke et al.,1994), by antigen-dependent killing (cytotoxic T lymphocyte assay,Tigges et al., 1996) or by cytokine secretion.

The presence of a cell-mediated immunological response can be determinedby proliferation assays (CD4 (+) T cells) or CTL (cytotoxic Tlymphocyte) assays. The relative contributions of humoral and cellularresponses to the protective or therapeutic effect of an immunogen can bedistinguished by separately isolating IgG and T-cells from an immunizedsyngeneic animal and measuring protective or therapeutic effect in asecond subject. As used herein and in the claims, the terms “antibody”or “immunoglobulin” are used interchangeably.

Optionally, an antibody or preferably an immunological portion of anantibody, can be chemically conjugated to, or expressed as, a fusionprotein with other proteins. For purposes of this specification and theaccompanying claims, all such fused proteins are included in thedefinition of antibodies or an immunological portion of an antibody.

In one embodiment a method includes treatment for a disease or conditioncaused by a HIV pathogen. In certain aspects embodiments include methodsof treatment of HIV infection infection, such as an infection acquiredfrom an HIV positive individual. In some embodiments, the treatment isadministered in the presence of HIV antigens. Furthermore, in someexamples, treatment comprises administration of other agents commonlyused against viral infection, such as one or more antiviral orantiretroviral compounds.

The therapeutic compositions are administered in a manner compatiblewith the dosage formulation, and in such amount as will betherapeutically effective. The quantity to be administered depends onthe subject to be treated. Precise amounts of active ingredient requiredto be administered depend on the judgment of the practitioner. Suitableregimes for initial administration and boosters are also variable, butare typified by an initial administration followed by subsequentadministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a polypeptide therapeutic are applicable.These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection and the like. The dosage of thecomposition will depend on the route of administration and will varyaccording to the size and health of the subject.

In certain instances, it will be desirable to have multipleadministrations of the composition, e.g., 2, 3, 4, 5, 6 or moreadministrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8,to 5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including all rangesthere between.

A. Combination Therapy

The compositions and related methods, particularly administration of anantibody that binds a DC receptor, e.g. CD40, and delivers an HIVantigen or a peptide to a patient/subject, may also be used incombination with the administration of traditional anti-retroviraltherapies. These include, but are not limited to, entry inhibitors, CCR5receptor antagonists, nucleoside reverse transcriptase inhibitors,nucleotide reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, protease inhibitors, integrase inhibitors andmaturation inhibitors.

In one aspect, it is contemplated that a therapy is used in conjunctionwith antiviral or anti-retroviral treatment. Alternatively, the therapymay precede or follow the other agent treatment by intervals rangingfrom minutes to weeks. In embodiments where the other agents and/or aproteins or polynucleotides are administered separately, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the therapeutic compositionwould still be able to exert an advantageously combined effect on thesubject. In such instances, it is contemplated that one may administerboth modalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for administration significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In yet another aspect, a vaccine may be administered as part of aprime/boost strategy. A priming vaccine dose can be administered usingan antibody fused to an HIV antigen in any of the embodiments describedherein. A vaccine boost can be administered through the use of a secondvaccine, either of the same type or from a different type of vaccine.Examples of such different vaccines include DNA vaccines (naked or not)or a recombinant poxvirus. A recombinant pox virus can be selected fromthe group comprising NYVAC, NYVAC-KC, ALVAC and MVA virus. In someembodiments, the second vaccine, such as a pox virus vaccine or a DNAvaccine is used in a priming dose and the vaccines of the disclosurecomprising the anti-DCR-HIV antigen fusion proteins, e.g theanti-CD40-HIV antigen fusion proteins, are administered in one or morebooster doses. In one embodiment, the DNA vaccine is selected fromDNA-HIV-PT123 DNA vaccine.

DNA-HIV-PT123 HIV vaccine may be presented as a solution for injectionat a total DNA concentration of approximately 4.0 mg/ml in PBS buffer.The vaccine is an equi-mass mixture of three different recombinantplasmids expressing clade C 96ZM651 gp140, 96ZM651 Gag, and 97CD54Pol-Nef. The DNA plasmid backbone was developed by the Vaccine ResearchCenter (VRC), NIAID. The CMV/R promoter consists of the translationalenhancer region of the CMV immediate early region 1 enhancer substitutedwith the 5′-untranslated human T cell leukemia virus type 1 (HTLV-1)R-U5 region of the long terminal repeat (LTR) to optimize geneexpression. Other elements of the plasmid include a bovine growthhormone polyadenylation signal termination sequence (Tbgh) and akanamycin resistance cassette (Kan). Enhancements made to the insertsinclude RNA and codon optimization, RNA secondary structure modulation,splice sites removal, TCF binding sites removal, and increasing the GCcontent.

The second vaccine may comprise additional HIV antigens apart from theEnv antigens that may be used in the first vaccine. It is alsocontemplated that the second vaccine may comprise an HIV protein such asan env protein plus an adjuvant either directly linked or administeredindependently.

Various combinations of therapy may be employed, for example antiviralor antiretroviral therapy is “A” and an antibody vaccine that comprisesan antibody that binds a DC receptor and delivers an HIV antigen or apeptide or consensus peptide thereof is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the antibody compositions to a patient/subject willfollow general protocols for the administration of such compounds,taking into account the toxicity, if any, of the composition. It isexpected that the treatment cycles would be repeated as necessary. It isalso contemplated that various standard therapies, such as hydration,may be applied in combination with the described therapy.

B. General Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects may involve administering an effective amountof a composition to a subject. In some embodiments, an antibody thatbinds DC receptor, e.g. CD40, and delivers an HIV antigen or a peptideor consensus peptide thereof may be administered to the patient toprotect against or treat infection by one or more HIV subtypes.Alternatively, an expression vector encoding one or more such antibodiesor polypeptides or peptides may be given to a patient as a preventativetreatment. Additionally, such compositions can be administered incombination with an antibiotic. Such compositions will generally bedissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-infective agents and vaccines, can also be incorporated into thecompositions.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the mucosal, intravenous,intradermal, intramuscular, sub-cutaneous, or even intraperitonealroutes. Typically, such compositions can be prepared as either liquidsolutions or suspensions; solid forms suitable for use to preparesolutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and, the preparations can also beemulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization or an equivalent procedure. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques, which yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, nasal, or buccaladministration. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal,or intravenous injection. In certain embodiments, a vaccine compositionmay be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specificallyincorporated by reference). Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

IV. LISTING OF SEQUENCES

SEQ ID NO Brief Description SEQUENCE 1 Amino acidNLWVTVYYGVPVWKEAKTTLFCASDAKSYEKEVHNVWATHAC sequence of EnvVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDQSLKP gp140CVKLTPLCVTLNCTEVNVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQSIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHFPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKTDIWDNMTWMQWDREISNYTNTIYRLLEDSQSQQEQNEKD LLALDSWNNLWNWFDITKWLWYIK 2Amino acid QTPTNTISVTPTNNSTPTNNSNPKPNP sequence of FlexV1 3 Amino acidasQTPTNTISVTPTNNSTPTNNSNPKPNPasNLWVTVYYGVPVWKEA sequence of JS-KTTLFCASDAKSYEKEVHNVWATHACVPTDPNPQEIVLGNVTEN FlexV1-JS-EnvFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTEVN gp140-JS; JSVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVY sites are lowerALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFD case; FlexV1 PIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVST is in italics; QLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQ Env gp140 is SIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHF underlinedPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKTDIWDNMTWMQWDREISNYTNTIYRLLEDSQSQQEQNEKDLLALDSWNNLWNWFD ITKWLWYIKas 4 Amino acidEVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKG sequence ofLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRA humanized heavyEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAP chain of anti-CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ CD40 antibodySSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG 12E12 (hAnti-PPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED CD40VH3-LV-PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW hIgG4HC)LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 5 Amino acidDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVK sequence ofLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFN humanized lightKLPPTFGGGTKLEIKGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF chain of anti-YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA CD40 antibodyDYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12E12 (hAnti- CD4OVH3-LV- hIgG4HC) 6Fusion EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKG polypeptide; JSLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRA sites are lowerEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAP case; FlexV1 is inCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ italics; Env gp140SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG is underlinedPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKasQTPTNTISVTPTNNSTPTNNSNPKPNPasNLWVTVYYGVPVWKEAKTTLFCASDAKVYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTEVNVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQSIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHFPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKTDIWDNMTWMQWDREISNYTNTIYRLLEDSQSQQEQNEKDLLALDSWNNLWNWFDITK WLWYIKas 7 Coding ntATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT sequence of SEQCCCAGGCGCGCGATGTGATATCCAGATGACACAGAGCCCTTCC ID NO: 5TCCCTGTCTGCCTCTGTGGGAGACAGAGTCACCATCACCTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGGCAAGGCCGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATACCCTCACCATCAGCTCCCTGCAGCCTGAAGATTTCGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAUCT(GAGATCAAAGGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGTTAG 8 Coding ntATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTAC sequence of fusionGCGTGTCCACTCCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGC polypeptide withTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCT signal sequenceCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAG (SEQ ID NO: 9)GCCCCAGGCAAGGGCCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAACAGCCTGAGGGCCGAGGACACAGCCGTGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCGAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCCAGACCCCGACCAACACCATTTCCGTGACCCCCACCAACAATAGCACTCCGACGAACAACAGCAACCCCAAGCCCAACCCGGCATCAAACCTCTG6GTGACCGTGTACTATGGCGTCCCTGTGTGGAAAGAAGCCAAGACCACCCTGTTCTGCGCGTCCGACGCCAAGGTCTACGAAAAGGAGGTGCACAATGTCTGGGCCACFCACGCCTGCGTCCCCACTGACCCAAACCCACAAGAAATCGTGCTGGGGAACGTGACCGAGAACTTCAATATGTGGAAGAACGACATGGTGGACCAGATGCATGAGGATATCATCAGCCTGTGGGACCAGTCGCTCAAGCCTTGCGTCAAGCTGACTCCTCTGTGTGTGACCTTGAACTGTACTGAAGTGAACGTGACCAGGAACGTCAACAACAGCGTGGTCAACAACACTACCAACGTGAACAAGTCCATGAACGGAGACATGAAGAATTGCTCCTTCAACATCACCACCGAACTCAAGGACAAGAAGAAGAATGTGTACGCCCTGTTCTACAAGTTGGACATCGTGTCCCTCAACGAAACTGACGATTCCGAAACCGGGAACTCGTCCAAGTATTACCGGCTCATCAACTGCAACACCTCCGCCCTGACTCAGGCTTGCCGAAAGTGTCCTTCGACCCAATTCCGATCCATTACTGCGCCCCCGCCGGTTACGCCATTCTGAAGTGCAACAATAAGACCTTCAACGGAACAGGCCCCTGCCACAACGTGTCGACCGTGCAGTGCACACACGGTATCAAACCCGTCGTGTCCACCCAACTCCTGCTGAACGGCTCACTGGCTGAGGAGGGTATTATCATCCGGTCCGAGAACCTGACTAACAACGTGAAAACCATTATCGTGCACCTGAACCGATCGATCGAAATCGTCTGCGTGCGCCCTAACAACAATACTCGGCAGTCCATCCGGATCGGGCCTGGACAGACTTTCTACGCGACCGGAGATATCATTGGAGATATCAGACAGGCGCACTGTAACATCTCCCGCACCAACTGGACCAAGACCCTGAGAGAAGTCAGAAACAAGCTCCGAGAGCACTTCCCCAACAAGAACATCACCTTTAAGCCGTCCTCCGGCGGCGACCTGGAGATTACCACTCATCGTTCAACTGCCGCGGGGAATTCTTCTACTGTAATACCTCCGGACTGTTTTCCATCAACTACACTGAAAACAACACCGATGGCACCCCGATTACCCTTCCGTGCCGGATTAGGCAGATCATTAATATGTGGCAGGAGGTCGGACGGGCTATGTACGCCCCGCCGATTGAGGGAAATATCGCCTGCAAATCCGACATTACTGGCCTGCTGCTCGTGCGCGACGGAGGCTCSACCAACGACAGCACCAACAACAACACTGAGATCTTCCGGCCCGCCGGCGGAGATATGAGAGATAACTGGAGGTCCGAACTTTACAAGTACAAGGTCGTGGAAATCAAGCCGCTTGGTATTGCACCTACCGAGGCCAAGAGAAGAGTGGTGGAGCGGGAGAAGCGGGCAGTGGGGATCGGAGCCGTGTTCCTGGGATTCCTGGGCGCGGCGGGCTCGACCATGGGAGCGGCCTCTATTACCCTGACGGCTCAGGCCCGCCAAGTGCTGAGCGGAATCGTGCAGCAGCAATCGAATCTGCTGCGGGCCATCGAAGCCCAGCAGCACCTCTTGCAACTTACTGTGTGGGGTATCAAGCAGCTTCAAACTCGCGTGTTGGCCATAGAACGCTACCTGAAGGACCAGCAGTTGCTCGGACTCTGGGGTTGCAGCGGGAAGCTGATTTGCACTACTGCCGTGCCGTGGAACATCTCCTGGTCAAACAAGAGCAAAACCGACATTTGGGACAACATGACGTGGATGCAGTGGGATCGSGAGATCTCAAACTACACTAACACCATCTACCGCCTGCTGGAGGACTCCCAGTCACAACAGGAACAGAACGAAAAGGATCTGCTGGCACTGGACTCATGGAACAACCTGTGGAACTGGTTTGACATCACCAAGTGGC TGTGGTACATCAAGGCGTCTTGA 9Fusion MGWSLILLFLVAVATRVHSEVQLVESGGGLVQPGGSLKLSCATSG polypeptide withFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTI signal peptideSRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGT Signal peptide isLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS double underlined;WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV JS sites are lowerDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL case; FlexV1 is inMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ italics; Env gp140FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK is underlinedGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKasQTPTNTISVTPTNNSTPTNNSNPKPNPasNLWVTVYYGVPVWKEAKTTLFCASDAKVYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTEVNVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQSIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHFPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLGLWGCSGKLICTTAVPWNISWSNKSKTDIWDNMTWMQWDREISNYTNTIYRLLEDSQSQQEQNEKDL LALDSWNNLWNWFDITKWLWYIKas10 (12E12) HCD40- GFTFSDYYMY CDR1H 11 (12E12) HCD40- YINSGGGSTYYPDTVKGCDR2H 12 (12E12) HCD40- RGLPFHAMDY CDR3H 13 (12E12) LCD40- SASQGISNYLNCDR1L 14 (12E12) LCD40- YTSILHS CDR2L 15 (12E12) LCD40- QQFNKLPPT CDR3L16 rAB- EVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKS pIRES2[manti-LEWIGRINPYNGATSYNQNFKDKASLTVDKSSSTAYMELHSLTSE CD40_11B6.1C3_DSAVYYCAREDYVYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSE H-LV-hIgG4H-C]STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS 17 rAB -DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKP pIRES2[manti-GQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFALKISRVEAEDLGVY CD40_11B6.1C3FCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV K-LV-hIgGK-C]VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 18 (11B6) HCD40- GYSFTGYYMH CDR1H19 (11B6) HCD40- RINPYNGATSYNQNFKD CDR2H 20 (11B6) HCD40- EDYVY CDR3H 21(11B6) LCD40- RSSQSLVHSNGNTYLH CDR1L 22 (11B6) LCD40- KVSNRFS CDR2L 23(11B6) LCD40- SQSTHVPWT CDR3L 24 rAB-EVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQ pIRES2[manti-GLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTS CD40_12B4.2C10_EDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAKTKGPSVFP H-LV-hIgG4H-C]LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKAS 25 rAB-DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVK pIRES2[manti-LLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGN CD40_12B4.2C10_TLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN K-LV-v2-FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK hIgGK-C]ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 (12B4) HCD40- GYTFTDYVLH CDR1H 27(12B4) HCD40- YINPYNDGTKYNEKFKG CDR2H 28 (12B4) HCD40- GYPAYSGYAMDYCDR3H 29 (12B4) LCD40- RASQDISNYLN CDR1L 30 (12B4) LCD40- YTSRLHS CDR2L31 (12B4) LCD40- HHGNTLPWT CDR3L

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Superiority in Rhesus Macaques of Targeting HIV-1 Env Gp140 toCD40 Versus LOX-1 in Combination with Replication Competent NYVAC-KC forInduction of Env-Specific Antibody and T Cell Responses

The inventors compared the HIV-1-specific immune responses generated bytargeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1,two endocytic receptors on dendritic cells (DCs), in Rhesus macaquesprimed with a poxvirus vector (NYVAC-KC) expressing Env gp140. TheDC-targeting vaccines, humanized recombinant monoclonal antibodies fusedto Env gp140, were administered as a boost with poly ICLC adjuvanteither alone or co-administered with the NYVAC-KC vector. All theDC-targeting vaccine administrations with poly ICLC increased thelow-level serum anti-Env IgG responses elicited by NYVAC-KC primingsignificantly more (up to P=0.01) than a group without poly ICLC. Theresponses were robust, cross-reactive, and contained antibodies specificto multiple epitopes within gp140 including the C1, C2, V1-3, C4, C5,and gp41 immuno-dominant regions. The DC-targeting vaccines alsoelicited modest serum Env-specific IgA responses. All groups gave serumneutralization activity limited to Tier 1 viruses and antibody dependentcytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore,CD4⁺ and CD8⁺ T cell responses specific to multiple Env epitopes werestrongly boosted by the DC-targeting vaccines+poly ICLC. Together, theseresults indicate that prime/boost immunization via NYVAC-KC and eitherαCD40.Env gp140/poly ICLC or αLOX-1.Env gp140/poly ICLC induced balancedantibody and T cell responses against HIV-1 Env. Co-administration ofNYVAC-KC with the DC-targeting vaccines increased T cell responses, buthad minimal effects on antibody responses except for suppressing serumIgA responses. Overall, compared to LOX-1, targeting Env to CD40 gavemore robust T cell and serum antibody responses with broader epitoperepresentation and greater durability.

In this study, we directly compare the efficacy of targeting HIV-1 Envgp140 to CD40 versus LOX-1 in combination with replication competentvaccinia virus vector NYVAC-KC encoding Env gp140. Specifically, thisstudy aims to: i) establish the safety and immunogenicity of NYVAC-KCprime followed by boost via DC-targeting to either LOX-1 or CD40formulated with poly ICLC adjuvant and either administered alone orco-administered with NYVAC-KC; ii) compare the boosting ability ofDC-targeting via either LOX-1 or CD40 formulated with poly ICLC adjuvanteither administered alone or co-administered with NYVAC-KC; and iii)test the boosting ability of DC-targeting via CD40 eitherco-administered with poly ICLC or administered alone.

A. Materials and Methods

1. Production and Quality Assurance of αCD40.Env Gp140 and αLOX-1.Envgp140.

αCD40.Env gp140 was derived from the parental anti-human CD40 12E12recombinant human IgG4 mAb (GenBank HQ738667.1 and HQ738666.1) viahumanization of the mouse variable regions (Antitope, Ltd) as defined bythe variable regions in GenBank KM660791 and KM660792. Env gp140sequence derived from the codon optimized HIV-1 96ZM651 syntheticconstruct (NIH AIDS reagent program, GenBank AY181197.1 residues94-2064) was inserted at the IgG4 heavy chain C-terminal codon distal toa flexV1 flexible linker spacer and proximal to 6 His codons. αLOX-1.Envgp140 was derived from the parental anti-human LOX-1 15C4 recombinanthuman IgG4 mAb; GenBank KM246787 and KM246788) via humanization of themouse variable regions (Antitope, Ltd). This was fused to Env gp140sequence as described above. Specificity and relative binding affinityof αCD40.Env gp140 and αLOX-1.Env gp140 to human and Rhesus macaque CD40and LOX-1 ectodomains (encoded respectively by GenBank gbIAAO43990.11residues 22-193, and refINP_001252791.11 residues 21-193,dbjlAB102861.11 residues 169-918 and refINM_001194668.11 residues295-945) fused via the cohesion C-terminus (GenBank gbICP000568.11residues 3622666-3623172 with a Nhe I site linker) was tested by anadaption of multiplexed bead-based assay for equilibrium competitionbinding analysis of αLOX-1 Env gp140 and αCD40 Env gp140 interactionwith human and NHP LOX-1 and CD40 ectodomains. In this assay, beads werecoated with human LOX-1, NHP LOX-1, human CD40, or NHP CD40 ectodomainsand incubated overnight with 10 ng/ml of the parental mouse αLOX-1 15C4or αCD40 12E12 mAbs and varying concentrations of humanized αLOX-1,αLOX-1 Env gp140, αCD40, or αCD40 Env gp140, then probed with PE-labeledanti-mouse IgG, and analyzed with a Bio-Plex 200 instrument. There wasno significant difference between the binding of humanized αLOX-1 vs.αLOX-1 Env gp140 to human (EC₅₀ 0.6 μM) or NHP LOX-1 (IC₅₀ 0.6 μM)coated beads, and the binding of humanized αCD40 vs. αCD40 Env gp140 tohuman (IC₅₀ 1 μM vs. 1.2 μM) or NHP (IC₅₀ 0.83 μM vs. 1.2 μM) CD40coated beads was similar (data not shown). The vaccines were formulatedand administered exactly as described below.

2. Animals and Assays.

Thirty male Rhesus macaques ranging in age from 3 to 6 years andweighing at least 4 kg were procured from Harlan Laboratories and housedat the Advanced Biosciences Laboratories (ABL) animal facility inRockville, Md. The ABL in vivo facility is USDA-registered andaccredited by the American Association for the Accreditation ofLaboratory Animal Care International (AAALAC). ABL's veterinarypractices comply with all policies of the “Guide for the Care and Use ofLaboratory Animals,” DHHS (NIH 85-23), Animal Welfare (DHHS-TN 73-2) theNIH Manual Issuance 4206 and 6000-3-4-58, “Responsibility for Care andUse of Animals CDC/NIH 4th edition”, “Biosafety in Microbiological andBiomedical Laboratories,” and Public Health Service Policy on HumaneCare and Use of Laboratory Animals under a Category 1 assurance fromOLAW. All procedures were carried out under Ketamine anesthesia bytrained personnel under the supervision of veterinary staff and allefforts were made to ameliorate the welfare and to minimize animalsuffering in accordance with the “Weatherall report for the use ofnon-human primates” recommendations. Other than for a seven-daypost-inoculation follow up observation period, animals were pair-housedin adjoining primate cages allowing social interactions, undercontrolled conditions of humidity, temperature and light (12-hourlight/12-hour dark cycles). Food and water were available ad libitum.Animals were monitored and fed standard laboratory rations twice daily.Trained personnel offered dietary supplements with fresh fruit andoccasional treats at least once a day. Early endpoint criteria, asproposed by the project team and approved by the IACUC, were used todetermine when animals should be humanely euthanized. The ABLveterinarian was authorized to determine whether animals met suchcriteria and if necessary, was tasked to stabilize any affected animalsprior to consulting with the lead investigators. ABL's InstitutionalAnimal Care and Use Committee (IACUC) approved of the proposed studyprotocol prior to the initiation of any in vivo work. The protocolnumber assigned by the IACUC/ethics committee that approved this studyis AUP567.

3. Vaccination Protocols.

The NYVAC-KC virus inoculum was provided in 0.2 ml aliquots as NYVAC-KCexpressing Env gp140 (ZM96) and ZM96 Gag plus CN54 PolNef at 0.1 ml ofeach virus @1.2×10⁸ pfu per vial. The immunogen mixture according tomolar ratios was Env:GagPolNef=1:1. Prior to administration, therequired number of vaccine vials containing the virus mixture werethawed at 37° C. and put on ice immediately after thawing. 1 ml of 1×TBSwas added to each vial and briefly vortexed. Sonication with a Bransonic1210 unit was performed according to the following procedure: fill waterbath of the sonicator with water and ice; sonicate sample 3 times for 10sec each time; after the sonication steps, vortex sample. For eachanimal, 1 ml of the vaccine preparation was drawn up from the vials by a1 ml syringe and kept on ice until administration. Before immunization,the skin of the upper arm (deltoid) was shaved and cleaned with alcohol.Animals received the vaccine preparation via intramuscular injection of1 ml of the vaccine preparation @2×10⁸ pfu/ml total of 2 viruses in thedeltoid. The poly ICLC was provided in 1 ml vials at a concentration of2 mg/ml. Vials (Hiltonol Lot: PJ215-1-10-01) were stored at 2-8° C.Administration was 1 mg (subcutaneous) in two injections of 250 μl inthe center of each circular injection pattern (3-4 cm diameter) formedby the intradermal administrations of αLOX-1.Env gp140 or αCD40.Envgp140 vaccine components which were stored in 1 M Arginine+100 mMTris.HCl Buffer pH 6-8. Protein vaccine administrations were at a 200 μgdose, intradermal in a total of 8 injections of 250 μl each (2 ml totalinjection)—four injections was performed in each side of the back placedin a circular pattern. To avoid toxicity of 1 M Arginine buffer, theconcentrated protein was diluted approximately 1:4 in PBS before use.Each animal received in the upper back (dorsal thoracic) a total of 8intradermal injections of 250 μl each. Four injections were performed ineach side of the back placed in a circular pattern of 3-4 cm ofdiameter. Skin was shaved before injection and cleaned with 70% alcoholsolution. Intradermal injections were performed using an insulinsyringe. The injection of poly ICLC was performed after the i.d.injections of the proteins and in the middle of each circle with 500 μlinjected s.c. This administration procedure was designed to promotedrainage of antigen and adjuvant to the same lymph node site and at therecommendation of ABL's head veterinarian. The supervising veterinarianreported no adverse events from the vaccinations.

4. Neutralization Assay.

Neutralizing antibodies were measured as a function of reductions inluciferase (Luc) reporter gene expression after a single round ofinfection in TZM-bl cells. TZM-bl cells (also called JC57BL-13) and A3R5cells were obtained from the NIH AIDS Research and Reference ReagentProgram, as contributed by John Kappes and Xiaoyun Wu. Briefly, apre-titrated dose of virus was incubated with serial 3-fold dilutions oftest sample in duplicate in a total volume of 150 μl for 1 h at 37° C.in 96-well flat-bottom culture plates. Freshly trypsinized cells (10,000cells in 100 μl of growth medium containing 75 μg/ml DEAE dextran) wereadded to each well. One set of 8 control wells received cells+virus(virus control) and another set received cells only (backgroundcontrol). After 48 hours of incubation, 100 μl of cells was transferredto a 96-well black solid plate (Costar) for measurements of luminescenceusing the Britelite Luminescence Reporter Gene Assay System (PerkinElmerLife Sciences). Assay stocks of molecularly cloned Env-pseudotypedviruses were prepared by transfection in 293T/17 cells (American TypeCulture Collection) and titrated in TZM-bl cells. This assay has beenformally optimized and validated, and was performed in compliance withGood Clinical Laboratory Practices (GCLP), including participation in aformal proficiency testing program. Additional information on the assayand all supporting protocols may be found on the world wide web at:hiv.lanl.gov/content/nab-reference-strains/html/home.htm. The method forMB analysis of serum neutralization assays as shown in FIG. 4.

5. HIV-1 Specific Binding Antibody Assay.

HIV-1 specific IgG antibodies to gp120/gp140 proteins and V1/V2scaffolds were measured by an HIV-1 binding antibody multiplex assay.All assays were run under GCLP compliant conditions, including trackingof positive controls by Levy-Jennings charts using 21CFR Part 11compliant software. Positive controls included a HIVIG and CH58 mAb IgGtitration. Negative controls included in every assay were blank(uncoupled) and MulVgp70 His6 (empty gp70 scaffold) coupled beads, ablank well on each assay plate, as well as HIV-1 negative sera. Tocontrol for antigen performance, we used the preset criteria that thepositive control titer (HIVIG) included on each assay (and for assayswith V1V2 antigens, CH58 mAb, had to be within +/−3 standard deviationsof the mean for each antigen (tracked with a Levy-Jennings plot withpreset acceptance of titer and calculated with a four-parameter logisticequation, SigmaPlot, Systat Software). Antibody measurements wereacquired on a Bio-Plex instrument (Bio-Rad, Hercules, Calif.) using21CFR Part 11 compliant software and the readout is in MFI. Thefollowing antigens were examined: ZM96 gp140-C tag (Baylor Health); (ConS gp140 CF (a group M consensus envelope gp140; A1.con.env03 140 CF(clade A Consensus), B.con.env03 140 CF (clade B Consensus), C.con.env03140 CF (clade C Consensus), clade A 00MSA 4076 gp140, gp70 control, gp70B.CaseA2 V1/V2 (a recombinant clade B gp70 scaffold protein with theV1V2 variable region), C.1086V1-V2 Tags and C.1086 V2 tags (atransmitted clade C isolate provided by Drs. Liao and Haynes, Duke) andAE.A244 V1/V2 tags, AE.A244 V2 tags (Clade AE sequence of RV144 vaccineimmunogen.

6. Serum Antibody Linear Epitope Mapping.

Serum epitope mapping of heterologous strains was performed aspreviously described with minor modifications. Briefly, array slideswere provided by JPT Peptide Technologies GmbH (Germany) by printing alibrary designed by Dr. B. Korber, Los Alamos National Laboratory, ontoEpoxy glass slides (PolyAn GmbH, Germany). The library containsoverlapping peptides (15-mers overlapping by 12) covering 6 full lengthgp160 consensus sequences (Clade A, B, C, D, Group M, CRF1, and CRF2),and gp120 sequences of 6 vaccine strains (MN, A244, Th023, TV-1, ZM651,1086C). 3 identical subarrays, each containing the full peptide library,were printed on each slide. All array slides were blocked for 1 hour,followed by a 2 hr incubation with 1:50 diluted serum samples and asubsequent 45 min incubation with Goat Anti-Hu IgG conjugated with AF647(Jackson ImmunoResearch, PA). Array slides were scanned at a wavelengthof 635 nm using an Axon Genepix 4300 Scanner (Molecular Devices,Sunnyvale, Calif., USA). Images were analyzed using Genepix Pro 7software (Molecular Devices) to obtain binding intensity values for allpeptides. Binding of postimmunization serum to each peptide wassubtracted of its own baseline value, which was defined as the mediansignal intensity of the triplicates of the peptide for the matchedprebleed serum plus 3 times the standard error of the triplicates.Binding magnitude to each identified epitope was defined as the highestbinding by a single peptide within the epitope region.

7. Intracellular Cytokine Staining.

Cryopreserved PBMC were thawed and rested overnight in R10/RPMI 1640(BioWhittaker, Walkersville, Md.), 10% FBS, 2 mM L-glutamine, 100 U/mlpenicillin G, 100 μg/ml streptomycin] with 50 U/ml Benzonase (Novagen,Madison, Wis.) in a 37° C./5% CO2 incubator. The following morning,cells were stimulated with peptide pools (2 μg/ml, described in 2) inthe presence of GogiPlug (10 μg/ml; BD Biosciences, San Jose, Calif.)for 6 h. Negative controls received an equal concentration of DMSOinstead of peptides. Subsequently, intracellular cytokine staining (ICS)was performed as described in the art. The following monoclonalantibodies were used: CD4-BV421 (clone OKT4; BioLegend), CD8-BV570(clone RPA-T8; BioLegend), CD69-ECD (clone TP1.55.3; Beckman Coulter),CD3-Cy7APC (clone SP34.2; BD Biosciences), IFN-γ-APC (clone B27; BDBiosciences), IL-2-PE (clone MQ1-17H12; BD Biosciences), and TNF-FITC(clone Mab11; BD Biosciences). Aqua LIVE/DEAD kit (Invitrogen, Carlsbad,Calif.) was used to exclude dead cells. All antibodies were previouslytitrated to determine the optimal concentration. Samples were acquiredon an LSR II flow cytometer and analyzed using FlowJo version 9.8(Treestar, Inc., Ashland, Oreg.).

8. ADCC

ADCC activity against MW965.1 gp120 (provided by the Duke CAVDrepository) coated CEM.NKR_(CCR5) (NIH AIDS Reagent Program, Division ofAIDS, NIAID, NIH: CEM.NKR-CCR5 from Dr. Alexandra Trkola) target cellswas measured using the ADCC-GranToxiLux (GTL) assay as previouslydescribed in the art. The peak activity is the maximum activity observedat any dilution and considered positive if above the 8% Granzyme Bactivity cut-off.

9. ELISPOT

Millipore 96 well filtration plates were pre-treated with 70% EtOH,washed 5× with 1×PBS and then coated with 5 μg/ml mouse-anti-human-IFNγantibody (BD Pharmingen) over night at 4° C. After blocking withcomplete RPMI for 2 h at 37° C., 2×10⁵ PBMCs (prepared as for ICS) werestimulated in triplicates with peptide pools at 1 μg/ml, or PHA (2.5μg/ml) as positive control, while addition of medium only served asnegative control. The peptides and peptide pools used are thosedescribed in the art. Plates were incubated at 37° C. for 18-24 h beforewashing with cold H₂O twice and PBS/T for five times. Biotinylatedanti-IFNγ-antibody (Mabtech) was added at 1 μg/ml for 1 hour at 37° C.and, after washing, a 1:2000 dilution of Avidin-HRP (VectorLaboratories) was added, again for 1 h at 37° C. After final washing,stable DAB (Invitrogen) was added for 2 min, and then the reaction wasstopped with water washing. After drying, the numbers of spots in eachwell were counted with an automated ELISPOT reader (CTL Immunospot).

10. Statistical Methods

Wilcoxon signed-rank tests or 2-tailed t tests are used to comparechange of marker value at specific time points with baseline within eachgroup. Comparison between groups at specific time points was made usingthe Wilcoxon rank sum test. Response rates were compared between groupsusing Fisher's exact test. Overall magnitude of ELISPOT response wascompared between groups G1 and G2 by fitting a random effect model usinglog transformed data measured after W14. A p-value of less than or equalto 0.05 is considered statistically significant. Statistical analyseswere done with R (version 3.1.2; The R foundation for StatisticalComputing, Vienna, Austria). For the antigen-specific antibodymeasurements several criteria are used to determine if data from anassay are acceptable and can be statistically analyzed. The blood drawrate must be within the allowable visit window as determined by theprotocol. Secondly, if the blank bead negative control exceeds 5,000MFI, the sample will be repeated. If the repeat value exceeds 5,000 MFI,the sample will be excluded from analysis due to high background. QC andstandard curve titers must fall within +/−3 standard deviations of thehistorical mean plotted on Levey Jennings charts. Sample and controlreplicates must also be within 20% CV. Samples are declared to havepositive responses if they meet three conditions: i) the MFI minus Blankbead or MulVgp70 His6 values are greater than or equal toantigen-specific cutoff (based on the average+3 standard deviations of60 seronegative plasma samples), ii) the MFI minus Blank values aregreater than 3 times the baseline MFI minus blank values, and iii) theMFI values are greater than 3 times the baseline MFI values. For eachantigen and visit, the magnitude of binding response among bothresponders and non-responders is compared between groups using theWilcoxon rank sum test. Response rates were compared between groupsusing Fisher's exact test. No adjustments were made for multiplecomparisons, as these are exploratory analyses for which increased Type1 error is tolerated for better sensitivity to detect effects. A p-valueof less than or equal to 0.05 is considered statistically significant.

B. Results

1. HIV-1 Env Gp140 Targeted to Either LOX-1 or CD40 Elicits Env-SpecificB Cell Responses in NHPs Primed with a Live Virus Vector.

The inventors previously described an anti-human LOX-1 recombinant humanIgG4 antibody fused via a flexible linker to the heavy (H) chainC-terminus to codon optimized Env gp140 protein from the clade C HIV-196ZM651 (called ZM96) strain (αLOX-1.Env gp140). A similar fusionprotein (αCD40.Env gp140) was generated linking gp140 to a humanizedanti-human CD40 recombinant human IgG4 antibody. αCD40.Env gp140retained specific binding to human and Rhesus macaque CD40 (seeMaterials and Methods). In the previous study αLOX-1.Env gp140administered intradermally (i.d.) with TLR3 ligand poly ICLC givennearby subcutaneously (s.c.) efficiently boosted both humoral andcellular responses in Rhesus macaques primed with two intramuscular(i.m.) injections of replication competent NYVAC-KC vaccinia virusvectors encoding HIV-1 Gag, Nef, Pol and Env gp140 sequences. To thisprotocol the inventors added other NHP groups to test the relativeefficacy of targeting Env gp140 via CD40 versus LOX-1, to investigatepotential benefits of co-administering NYVAC-KC viruses coding for Envgp140 and Gag, Nef, Pol with the DC-targeting vaccinations, and toestablish the potency of CD40 targeting in the absence of TLR3stimulation. Table 1 shows the overall study design and tissue samplingschedule.

In this study, Env-specific serum IgG responses were observed in allanimals at all 3 post-DC-targeting vaccination time points, includinghigh titers against the vaccine strain ZM96 gp140 FIG. 1A) as well asgroup M consensus (FIG. 1B, con S gp140 CF), clade C (FIG. 1C,C.con.env03 140 CF), and clade B (FIG. 1D, B.con.env03 140 CF) crossclade sequences. Titers in response to the two NYVAC-KC vaccinations(week 6) were modest, but were boosted in all groups to almost maximallevels after one DC-targeting vaccine administration. These responseswere maximal after the second DC-targeting vaccine administration (week26, the primary immunogenicity endpoint) and waned somewhat 8 weeksafter the final vaccinations (FIG. 1). IgG binding antibody responses toV1V2 region antigens (i.e., a response that correlated with decreasedrisk of HIV-1 infection in RV144 human clinical trial) were observed inonly a few NHPs at the early time point, but were boosted or evoked toalmost maximal levels by a single LOX-1 or CD40-targeting vaccination(FIG. 1E-H). Notably, IgG responses to V1V2 were significantly higher(p=0.01 week 26 and p=0.02 week 32) when NYVAC-KC was co-administeredwith CD40 but not LOX-1 targeting despite similar overall bindingantibody levels between the paired groups (see G1/G2, FIG. 1, Table 2).The inventors next confirmed that the V1V2 response in this study was inpart due to recognition of the V2 sequence (and not just C1-V1)contained within these antigens by demonstrating that all groupselicited responses to an antigen designed to only expose V2 regionconformational and linear epitopes (V2 Tags) (FIG. 1, Table 2). In allcases after the DC-targeting vaccinations, G4 N2Cp2 values weresignificantly higher than G5 N2C2 for all gp140 antigens and the gp70 BCaseA V1V2 antigen (p-value=0.01) indicating a significant benefit toco-administering poly ICLC with the CD40-targeting vaccine (Table 2).

Low levels of serum IgA specific to Env gp140 was detected in a fewanimals after the NYVAC-KC vaccinations, but was boosted most in G4N2Cp2 when viewed as response rate (FIG. 2).

Analysis of the breadth and magnitude of binding antibodies to linearepitopes was evaluated by peptide microarray mapping. Overall, theanimals developed binding antibodies against gp120 linear epitopes C1,C2, V3, C3, C4, and C5, with V3 dominating the responses (FIG. 3).Linear binding responses were overall cross-clades, with a preferencefor Clade C sequences. The C5.2 response was focused on Clade C and theC3 response was restricted to C.ZM651, while the V3 and C1.2 binding wasbroader cross-clades (Table 3). The magnitude of binding trended higherin Groups G2 N2[CpN]2 and G4 N2Cp2 compared to Groups G1 N2[LpN]2, G3N2Lp2 and G5 N2C25.

Analysis of neutralizing activity in week 14, week 26, and week 32plasma samples was performed via TZM.bl and A3R5 assays against a panelof Tier 1 and Tier 2 HIV-1 Env pseudotyped viruses. Neutralizingactivity was detected against MW965.26 (clade C, Tier 1A, FIG. 4A) andTH023.6 (CRF01 AE, Tier 1A, not shown). Little or no neutralization wasdetected against Ce1086_B2, Ce1176_A3, C32010_F5, and DU151.2 viruses(data not shown). Also, no neutralization was detected against96ZM651.2, Bal.26, CE1086_B2, MN.3, SF162.LS, TV1.21 or MLV (the controlfor nonspecific activity) (data not shown). Neutralization (NAb) titersagainst the sensitive viruses (MW965.26 and TH023.6) decreased somewhatfrom week 26 to week 32 in all groups. Group 4 N2Cp2 had significantlyhigher ID₅₀ titers to the sensitive viruses than other groups, as wellas significantly higher ID₅0 titers to MW965.26 than groups G1 N2(LpN)2and G5 (N2C2), at weeks 14 and 26 (Table 4). At week 32, 8 weeks postcompletion of all vaccinations, G3 N2Lp2 and G4 N2Cp2 had the highestneutralizing scores based on individual-specific and group-specificmagnitude-breadth (MB) analysis which measures the overall magnitude andbreadth of NAb activities for each individual time point (FIG. 4B).

Analysis of ADCC-mediated antibody responses in plasma samples collectedat week −4, 14, 26, and 32 were measured via GranToxLux (GTL) assay. Inall groups positive GTL responses were observed at week 14 and week 26,two weeks after the DC-targeting vaccine boosts. Group 4 (G4 N2Cp2)included more responses than other groups and positive GTL responses atweek 32 were only observed in groups G2 N2(CpN)2 and G4 N2Cp2 (FIG. 5).

2. HIV-1 Env Gp140 Targeted to Either LOX-1 or CD40 Elicits Env-SpecificT Cell Responses in NHPs Primed with a Live Virus Vector.

Peripheral HIV-1 antigen-specific T cell responses to the vaccinationswere evaluated by IFNγ ELISPOT analysis of PBMCs. Few or no responseswere detected against Env gp140 antigen after the NYVAC-KCadministrations, however the DC-targeting vaccine boost raisedsignificant responses against Env gp140 in all animals in all groupsexcept two of the six animals in G5 N2C2, and this included responsesagainst all three Env subdomains sampled (FIG. 6). No significantresponses were detected against other HIV-1 antigens represented in theNYVAC-KC Gag Nef Pol vector at any time points (not shown). In thepairwise comparisons G1 N2[LpN]2 vs. G3 N2Lp2 (i.e., LOX-1 targetingwith and without co-administered NYVAC-KC) there were no significantdifferences in anti-Env T cell responses at weeks 26 or 32 (FIG. 6).However, in the pairwise comparisons G2 N2[CpN]2 vs. G4 N2Cp2 (i.e.,CD40 targeting with and without co-administered NYVAC-KC) there were wasa trend towards higher response with co-administered NYVAC-KC at week 26(p=0.065) which became a significant difference (p=0.009) at week 32(FIG. 6). In the pairwise comparison G4 N2Cp2 vs. G5 N2C2 (i.e., CD40targeting with and without poly ICLC adjuvant) there was a trend tohigher responses with poly ICLC (p=0.143 with 6/6 vs. 2/6 responders) atweek 26, which became significant at week 32 (p=0.010 with 6/6 vs. 4/6responders) (FIG. 6).

Peripheral HIV-1 antigen-specific CD4⁺ T cell responses to theDC-targeting boost vaccinations (week 26 and 32) were also evaluated byintracellular cytokine staining (ICS) analysis of PBMCs. Low levelresponses were detected in some animals against HIV-1 antigensrepresented in the NYVAC-KC Gag Nef Pol vector at these sample times,but responses against Env antigens were higher in all groups and werepositive in almost all animals (FIG. 7). In pairwise comparisons, therewas a significant difference among responders to Env only between G4(N2Cp2) and G5 (N2C2) (i.e., CD40 targeting with versus without polyICLC adjuvant) at week 26 but not week 32 (p=0.004 and p=0.052;Mann-Whitney U P values). The CD4⁺ T cell responses elicited by theDC-targeting vaccines were of a high quality as IFNγ, IL-2, and TNFαwere detected in response to Env antigens (FIG. 7).

Peripheral HIV-1 antigen-specific CD8⁺ T cell responses to theDC-targeting boost vaccinations (week 26 and 32) were also evaluated byICS analysis of PBMCs. Low level responses were detected in some animalsagainst HIV-1 antigens represented in the NYVAC-KC Gag Nef Pol vector atthese sample times, but responses against Env antigens were generallyhigher for all groups (FIG. 8). In pairwise comparisons, there was asignificant difference among responders to any protein only with G2N2(CpN)2>G4 N2Cp2 at week 32 (p=0.029 Mann-Whitney U P value). Highquality CD8⁺ T cell responses (as measured by positive responders forIFNγ, IL-2, and TNFα) were especially evident in G2 N2(CpN)2 (FIG. 8).Unlike for CD4⁺ T cell responses, poly ICLC adjuvant did not benefit thedevelopment of Env-specific CD8⁺ T cell responses (see G4 (N2Cp2) versusG5 (N2C2), FIG. 8).

3. αCD40.Env Gp140 Fusion Protein Elicits a More Durable BindingAntibody Response Compared to αLOX-1.Env Gp140 Fusion Protein.

Durability of binding and neutralizing antibody responses were assessedby proportion of change per week, which was calculated as [(response atdurability time point (wk32—response at peak time point (wk26))/responseat peak time point (wk26)]/number of weeks between time points].Durability was not evaluated for ADCC and T cell response (ICS) due tolimited number of responders at the durability time point. Significantdifferences were observed in the rate of binding antibody responsedecline observed among the 5 groups of the current study (FIG. 9). Bothgroups with the αCD40.Env gp140 fusion protein trended for betterbinding response durability compared to the groups with αLOX-1.Env gp140fusion protein, i.e., G2 N2[CpN]2>G1 N2[LpN]2 (trend) and G4 N2Cp2>G3N2Lp2 (trend) for proportion of change per week (FIG. 9A). G4 N2Cp2showed the best durability among the groups, with significantly slowerdecline of responses compared to G1 N2[LpN]2 and G5 N2C2 for binding tomost gp140s including Con S gp140 CFI and C.con.env03 140 CF (P values0.0056-0.018, 2-tailed t test) (Table 5). Durability for neutralizationresponse was evaluated only for neutralization of MW965.26 (in TZM-blcells), due to limited positive responders for other viruses at thedurability time point. G4 N2Cp2 also trended for better neutralizingresponse durability compared to G3 N2Lp2 (FIG. 9B). No significantdifference was observed among the groups for the rate of neutralizationresponse decline (Table 5).

4. DC-Targeting Alters Both Humoral and Cellular Responses Compared to aNon-DC Targeted Env Boosting Regimen

The current study did not have a control arm with non-DC-targeted Envgp140. However, a recent study (Garcia-Arriaza J, et al., J Virol89:8525-8539) included a group of Rhesus macaques (called group 1 N2NP2(C) in Garcia-Arriaza et al.; here called ExtNDC-N2[NP]2) that receivedthe same 2 NYVAC-KC priming immunizations as in the current study, butboosted with non-DC-targeted Env gp120 (TV1 gp120+1086 gp120 in MF59,100 μg total) administered together with NYVAC-KC at weeks 12 and 24.Also relevant is another recent study (Zurawski G, et al., PLoS One11:e0153484) that included a group with the same 2 NYVAC-KC primingimmunizations as in the current study, and boosted with the sameαLOX-1.Env gp140+poly ICLC vaccine as the current study (called group 1in 2; here called ExtDC-N2Lp3), except dosing of αLOX-1.Env gp140 wasthree times at weeks 12, 16, and 20 vs. twice at weeks 12 and 24 in thecurrent study. Similar BAMA, neutralization, ADCC and ICS assays by thesame laboratories were performed on those samples as were done for thecurrent study.

Therefore, we were able to compare immunogenicity data for the currentstudy with data from the ExtNDC-N2[NP]2 and ExtDC-N2Lp3 immunizationanimals to gauge the effects of DC-targeting on the magnitude anddurability of antibody responses and on the cellular response profiles,with the caveats that the adjuvant (i.e., MF59 vs. poly ICLC) and theantigens (i.e., gp120 TV1 gp120+1086 gp120 vs. gp140 Z96M) weredifferent in the non-DC-targeted ExtNDC-N2[NP]2, and an additional doseof DC targeting vaccine was given in ExtDC-N2Lp3. These comparisonsrevealed that the non-DC-targeted Env (gp120) ExtNDC-N2[NP]2immunization elicited considerably higher binding, neutralizing, andADCC responses compared to the DC-targeting regimens in the currentstudy at the peak immunity time point (week 26 for both studies). Bothplasma IgG and IgA binding responses were significantly higher for theExtNDC-N2[NP]2 group at week 26 (FIG. 10A; A Table 6). However, when thepeak immunity time for ExtDC-N2Lp3 (same vaccine components as G3 ofcurrent study) was included in the analysis these differences wereminimized. Binding response, and especially the IgA response, wassignificantly higher in the DCtargeting ExtDC-N2Lp3 group than G3,indicating that a third DC-targeting boost is advantageous (FIG. 10A; ATable 6). Similarly, neutralizing ID50 titers measured on TZM-bl cellsand A3R5 cells against diverse virus targets were significantly higherin the ExtNDC-N2[NP]2 group than present study groups, although againthe DC-targeting ExtDC-N2Lp3 group developed significantly higherneutralizing responses against two clade C strains TV1 and MW965 (FIG.10B and Table 7), indicating an improvement of neutralizing response bya third boost. ADCC response measured with Env coated cells wassignificantly higher for the non-DC-targeted ExtNDC-N2[NP]2 compared toall 5 groups in the current study at week 26 and to the ExtDC-N2Lp3(FIG. 10C and A Table 8). Despite the significantly higher antibodyresponses in the non-DC-targeting ExtNDCN2[NP]2 group compared to all 5groups in the current study, differences in the levels of HIV-specificCD4+ and CD8+ T cell cytokine secreting responses were not assignificant (FIG. 10D). The proportions of CD4+ T cells that express anyof the 3 cytokines tested for the ExtNDC-N2[NP]2 was comparable with theG1 (for any HIV antigen stimulation), G2, and G4 groups in current study(Table 9), though significantly higher than G3, G5 and ExtDC groups(P<0.0001 to 0.046). Meanwhile, G2 and the ExtNDC groups showed CD8+ Tcell response comparable to that of the ExtDN group upon any Env antigenstimulation, and all groups from current study, and the ExtDC group,developed CD8+ T cell responses comparable to that of the ExtNDC groupupon any Env stimulation (Table 9). In addition, the ExtDC groupdeveloped T cell responses significantly higher than G3 group in currentstudy except for CD4+ response upon any Env stimulation, demonstratingthe beneficial effect of the additional boost for T cell response aswell. Durability for the external non-DC-targeted Env gp120 group(Ext-N2[NP]2) group was also evaluated. Durability time point was notavailable for the ExtDC-N2Lp3 group in these same assays, and thereforedurability was not analyzed for that group. The peak time point was alsoweek 26 for ExtNDC-N2[NP]2, whereas the durability time point was week36 (12 weeks post the 4th immunization) instead of week 32 in currentstudy. Comparison of proportion of change per week revealed generallybetter durability of binding and neutralizing responses in theExtNDC-N2[NP]2 (FIG. 11; Tables 10 and 11). ExtNDC group showedsignificantly better durability compared to G1 N2[LpN]2 for all gp140stested, significantly higher than G3 N2Lp2 and G5 N2C2 for most of thegp140s tested (P values ranged from <0.0001 to 0.034, 2-tailed t-test)(Table 10), and significantly higher durability for binding togp70.B.CaseA V1V2 scaffold compared to G1 N2[LpN]2 and G3 N2Lp2(P=0.007). However, no significant difference was found betweenExtNDC-N2[NP]2 and either G2 N2[CpN]2 or G4 N2Cp2 for binding to anyantigens (FIG. 11, Table 10), reflecting the advantage of CD40 targetingfor durability noted above. Durability of neutralizing response(proportion of change per week) for G2 N2[CpN]2 and G3 N2Lp2 compared tothe non-DC-targeting group was not significantly different, butExtNDC-N2[NP]2 was higher compared to G1 N2[LpN]2, G4 N2Cp2, G5 N2C2,and ExtDC groups (FIG. 11, Table 11). One caveat of comparing thedurability of DC-targeting groups in the current study and thenon-DC-targeting ExtNDC-N2[NP]2 by proportion of change per week was theunequal time (weeks) between the peak and durability time points of the2 studies. It has been shown that the decline of antibody responsesafter vaccination is at different rates over time, with a much fasterinitial decline (36). The current study had only 6 weeks between the 2time points, whereas in the ExtNDC-N2[NP]2 study there was a 10 weeksinterval. It is possible that by calculating proportion of change perweek, the durability analysis gave the non-DC-targeted ExtNDC-N2[NP]2 abiased advantage; while the durability of the DC-targeting groups in thecurrent study relative to the non-DCtargeting ExtNDC-N2[NP]2 could infact be higher.

C. Tables

TABLE 2 Comparison between paired groups for plasma IgG bindingmagnitudes. Antigen Group Antigen Week Group2-1 Group3-1 Group4-2Group4-3 Group5-4 gp140 00MSA 4076 gp140 26 4144 (0.18) 5109 (0.03*)8073 (0.24) 7108 (0.48) −14514 (<0.01**) 32 3358 (0.09) 2252 (0.03*)3866 (0.31) 4971 (0.24) −7798 (<0.01**) gp140 A1.con.env03 140 CF 263351 (0.18) 3788 (0.13) 4927 (0.18) 4490 (0.04*) −14802 (<0.01**) 323331 (0.09) 3464 (0.06) 6745 (0.18) 6612 (0.06) −11775 (<0.01**) gp140B.con.env03 140 CF 26 7901 (0.24) 1156 (0.48) −1150 (1) 5595 (0.24)−15361 (<0.01**) 32 2943 (0.18) 1360 (0.31) 2417 (0.7) 4000 (0.04*)−7386 (<0.01**) gp140 C.con.env03 140 CF avi 26 3404 (0.13) 1347 (0.82)4133 (0.31) 6190 (0.18) −10920 (<0.01**) 32 1694 (0.18) 1502 (0.31) 6995(0.24) 7187 (0.03*) −11436 (<0.01**) gp140 Con S gp140 CFI 26 5861(0.24) 2225 (0.24) −1879 (0.7) 1757 (0.39) −13036 (<0.01**) 32 2517(0.31) 3443 (0.13) 9369 (0.39) 8444 (0.18) −17893 (<0.01**) gp140 ZM96gp140-Ctag 26 9319 (0.09) 2786 (0.48) 4198 (0.24) 10731 (0.13) −23761(<0.01**) 32 7596 (0.09) 5197 (0.59) 9296 (0.31) 11695 (0.03*) −25617(<0.01**) V1V2 AE.A244 V1V2 26 2 (0.59) 2 (0.59) −210 (0.09) −210 (0.13)−9 (0.7) 32 −4 (0.87) 10 (0.63) 0 (0.69) −14 (0.81) −1 (0.94) V1V2AE.A244 V2 tags 293F 26 373 (0.18) −14 (0.82) −444 (0.13) −57 (0.69) 4(0.94) 32 43 (0.42) 9 (0.7) −45 (0.38) −11 (0.57) 12 (0.57) V1V2 C.1086V2 tags 293F 26 −5 (0.82) 19 (0.94) −20 (0.75) −45 (0.75) 3 (0.82) 32 24(0.87) 36 (0.59) −11 (0.63) −22 (0.09) −12 (0.87) V1V2 C.1086C V1 V2Tags 26 10284 (0.13) 2331 (0.7) −11860 (0.06) −3906 (0.39) −1500 (0.39)32 2843 (0.13) 741 (0.59) −2394 (0.39) −291 (1) −1419 (0.31) V1V2 gp70B.CaseA V1 V2 26 7836 (<0.01**) 2879 (0.18) −708 (0.7) 4249 (0.39) −8507(0.02*) 32 2318 (0.02*) 809 (0.18) 2951 (0.24) 4460 (0.04*) −5629(<0.01**)Test results shown are median difference between groups followed by pvalue in parentheses [median of differences (p values)]. P values arefrom Wilcoxon rank sum test. Values for comparison are AUC values asmeasured in BAMA (Materials and Methods) and shown in FIG. 1. Groups areG1 N2[LpN]2, G2 N2[CpN]2, G3 N2Lp2, G4 N2Cp2 and G5 N2C2 (Table 1).Significant differences are bolded and indicated by * for p<0.05 and **for p<0.01.

TABLE 3 Binding magnitude to linear epitopes by plasma IgG from Group1-5 animals at the peak response time point (week 26). C1.1 C1.2 C2 V3C3 C4 V5-C5 C5.2 gp41 ID Group Boost Animal 23-27 33-36 65-68 95-106111-112 135-139 148-152 157-162 187-189 1 NYVAC-KC + R734 97 2,830 2024,616 2,686 1 573 1,139 1 aLox/PolyICLC R745 54 3,567 154 14,430 1 12,838 269 1 R758 1 4,582 10,900 47,715 3,074 1,747 565 3,937 1 2NYVAC-KC + R730 1 17,417 7,308 5,314 125 151 1 45,165 1 aLox/PolyICLCR746 6,411 41,288 15,138 51,380 55,490 1,779 32,849 61,527 24,886 R7552,311 1 17,920 36,921 13,187 1 5,040 133 1 R756 1 10,337 3,976 61,649537 1 579 1,728 377 3 aLox/PoluICLC R731 1 482 40,485 22,175 3,355 3,66139 20,533 1 R739 1 147 1,030 1,056 1,013 733 14,977 6,623 1 R749 8781,194 4,168 3,796 17,472 13 509 43,939 1 4 aCD40/PolyICLC R736 20910,941 12,794 21,838 19,495 1,578 1,960 8,829 1 R741 2,821 1 3,80938,028 6,963 1 15 795 1 R748 65,300 14,777 7,002 31,993 9,670 2,7058,433 8,244 682 R754 15,305 2365 3,304 65,297 49,294 497 2,339 6,11517,375 5 aCD40 R732 1,820 189 4,235 12,947 1 5,352 1 5,218 1 R737 243355 8,480 4,639 3,836 2,574 50 1 1 R759 1 821 1 62,969 1,046 1 168 1 1Epitope regions for gp120 are as shown graphically in FIG. 3 and hereinclude data for the gp41 immunodominant (ID) region. Magnitude shownhere are maximum binding=per epitope, which is calculated as the highestsignal intensity to a single peptide within a given epitope region. Datain the table are bolded with higher values in bold. The rank order inhigher values was G4 N2Cp2 (n=16), G2 N2[CpN]2 (n=14), G3 N2Lp2 (n=6),G1 N2[LpN]2/G5 N2C2 (n=3).

TABLE 4 Comparison between paired groups for serum neutralizingresponses. Comparison Assay Type Isolate Week. 14 Week. 26 Week. 32Grp2-1 TZM-bl t.mw965 130 (0.037*) 129 (0.261) 27 (0.145) t.th023 0(0.462) −16 (1) 0 (0.462) Grp3-1 TZM-bl t.mw965 98 (0.199) 42 (1) 0 (1)t.th023 0 (0.462) −16 (0.655) 0 (0.405) Grp4-1 TZM-bl t.mw965 372(0.005*) 130 (0.045*) 68 (0.104) t.th023 0 (0.599) 20 (0.561) 0 (0.462)Grp5-1 TZM-bl t.mw965 35 (0.199) −32 (0.173) 0 (0.753) t.th023 0 (0.599)−16 (0.074) 0 (0.405) Grp3-2 TZM-bl t.mw965 −32 (0.81) −86 (0.378) −27(0.266) t.th023 0 (0.775) 0 (1) 0 (0.176) Grp4-2 TZM-bl t.mw965 242(0.093) 1 (0.378) 41 (0.683) t.th023 0 (1) 36 (0.798) 0 (1) Grp5-2TZM-bl t.mw965 −94 (0.128) −160 (0.045*) −27 (0.18) t.th023 0 (1) 0(0.176) 0 (0.176) Grp4-3 TZM-bl t.mw965 274 (0.173) 88 (0.092) 68(0.146) t.th023 0 (0.924) 36 (0.347) 0 (0.176) Grp5-3 TZM-bl t.mw965 −62(0.471) −74 (0.687) 0 (0.753) t.th023 0 (0.924) 0 (0.176) 0 (NaN) Grp5-4TZM-bl t.mw965 −336 (0.02*) −162 (0.031*) −68 (0.181) t.th023 0 (1) −36(0.028*) 0 (0.176)Test results shown are median difference between groups followed by pvalue in parentheses [median of differences (p values)]. Analysis was byt-test assuming unequal variance between groups and calculated P valuesare indicated in parentheses. Values used for comparison are serum ID₅₀titers as shown in FIG. 4. Significant differences (p<0.05) are boldedand indicated by *. Groups are G1 N2[LpN]2, G2 N2[CpN]2, G3 N2Lp2, G4N2Cp2 and G5 N2C2.

TABLE 5 Analysis of paired comparisons of serum ID₅₀ values. ResponseTested Comparison Magnitude Stats p Value IgG: G1 vs. G2 −0.091(0.028)vs. −0.045(0.067) 0.168 C.con.env03 G1 vs. G3 −0.091(0.028) vs.−0.069(0.029) 0.214 140 CF) G1 vs. G4 −0.091(0.028) vs. −0.041(0.014)0.006** G1 vs. G5 −0.091(0.028) vs. −0.092(0.035) 0.930 G2 vs. G3−0.045(0.067) vs. −0.069(0.029) 0.447 G2 vs. G4 −0.045(0.067) vs.−0.041(0.014) 0.907 G2 vs. G5 −0.045(0.067) vs. −0.092(0.035) 0.165 G3vs. G4 −0.069(0.029) vs. −0.041(0.014) 0.073 G3 vs. G5 −0.069(0.029) vs.−0.092(0.035) 0.236 G4 vs. G5 −0.041(0.014) vs. −0.092(0.035) 0.0142*IgG: Con S G1 vs. G2 −0.097(0.025) vs. −0.057(0.061) 0.180 gp140 CFI) G1vs. G3 −0.097(0.025) vs. −0.075(0.024) 0.153 G1 vs. G4 −0.097(0.025) vs.−0.052(0.031) 0.0182* G1 vs. G5 −0.097(0.025) vs. −0.108(0.028) 0.488 G2vs. G3 −0.057(0.061) vs. −0.075(0.024) 0.519 G2 vs. G4 −0.057(0.061) vs.−0.052(0.031) 0.856 G2 vs. G5 −0.057(0.061) vs. −0.108(0.028) 0.103 G3vs. G4 −0.075(0.024) vs. −0.052(0.031) 0.171 G3 vs. G5 −0.075(0.024) vs.−0.108(0.028) 0.054 G4 vs. G5 −0.052(0.031) vs. −0.108(0.028) 0.007**IgG: G1 vs. G2 −0.139(0.036) vs. −0.107(0.034) 0.153 gp70.B.CaseA G1 vs.G3 −0.139(0.036) vs. −0.125(0.029) 0.492 V1.V2) G1 vs. G4 −0.139(0.036)vs. −0.064(0.081) 0.081 G1 vs. G5 −0.139(0.036) vs. −0.142(0.041) 0.915G2 vs. G3 −0.107(0.034) vs. −0.125(0.029) 0.352 G2 vs. G4 −0.107(0.034)vs. −0.064(0.081) 0.272 G2 vs. G5 −0.107(0.034) vs. −0.142(0.041) 0.292G3 vs. G4 −0.125(0.029) vs. −0.064(0.081) 0.134 G3 vs. G5 −0.125(0.029)vs. −0.142(0.041) 0.575 G4 vs. G5 −0.064(0.081) vs. −0.142(0.041) 0.101Neutralization: G1 vs. G2  −0.14(0.007) vs. −0.116(0.054) 0.327 MW965.26G1 vs. G3  −0.14(0.007) vs. −0.136(0.031) 0.790 G1 vs. G4  −0.14(0.007)vs. −0.124(0.026) 0.184 G1 vs. G5  −0.14(0.007) vs. −0.122(0.018) 0.088G2 vs. G3 −0.116(0.054) vs. −0.136(0.031) 0.486 G2 vs. G4 −0.116(0.054)vs. −0.124(0.026) 0.760 G2 vs. G5 −0.116(0.054) vs. −0.122(0.018) 0.817G3 vs. G4 −0.136(0.031) vs. −0.124(0.026) 0.546 G3 vs. G5 −0.136(0.031)vs. −0.122(0.018) 0.461 G4 vs. G5 −0.124(0.026) vs. −0.122(0.018) 0.881Test results shown are median difference between groups followed by pvalue in parentheses [median of differences (p values)]. Analysis was byt-test assuming unequal variance between groups and calculated P valuesare indicated in parentheses. Values used for comparison are plasmabinding AUC (as shown in FIG. 3) and serum neutralization ID₅₀ titersmeasured on TZM-bl cells (as shown in FIG. 4). Groups are G1 N2[LpN]2,G2 N2[CpN]2, G3 N2Lp2, G4

N2Cp2 and G5 N2C2 (Table 1). Significant differences are bolded andindicated by * for p<0.05 and ** for p<0.01.

TABLE 6 BAMA peak time point IgG (AUC) and IgA (MFI) magnitude pairwisecomparison of non-DC targeting ExtNDC-N2[NP]2 against DC-targeted groups(G1 to G5 and ExtDC in the current study and ExtDC-N2Lp3) as well asExtDC against G3. Isotype Analyte Comparison Mean (SD) p Value IgG 00MSA4076 gp140 ExtNDC vs. G1 29184(9567) vs. 5334(4248)  p = 0.0567 IgG00MSA 4076 gp140 ExtNDC vs. G2 29184(9567) vs. 11097(7232) p = 0.0099IgG 00MSA 4076 gp140 ExtNDC vs. G3 29184(9567) vs. 11454(4469) p =0.0006 IgG 00MSA 4076 gp140 ExtNDC vs. G4 29184(9567) vs. 14785(4803) p= 0.0078 IgG 00MSA 4076 gp140 ExtNDC vs. G5 29184(9567) vs. 2729(1299) p < 0.0001 IgG 00MSA 4076 gp140 ExtNDC vs. ExtDC 29184(9567) vs.16819(5849) p = 0.016 IgG 00MSA 4076 gp140 G3 vs. ExtDC 11454(4470) vs.16819(5850) p = 0.0934 IgG A1.con.env03 140 CF ExtNDC vs. G1 39323(8491)vs. 11416(4205) p = 0.0002 IgG A1.con.env03 140 CF ExtNDC vs. G239323(8491) vs. 16951(7958) p = 0.0022 IgG A1.con.env03 140 CF ExtNDCvs. G3 39323(8491) vs. 14794(3346) p < 0.0001 IgG A1.con.env03 140 CFExtNDC vs. G4 39323(8491) vs. 21629(5953) p = 0.001 IgG A1.con.env03 140CF ExtNDC vs. G5 39323(8491) vs. 6165(4560)  p = 0.0006 IgG A1.con.env03140 CF ExtNDC vs. ExtDC 39323(8491) vs. 30388(9128) p = 0.1032 IgGA1.con.env03 140 CF G3 vs. ExtDC  14794(3346 vs. 30388(9128) p = 0.002IgG B.con.env03 140 CF ExtNDC vs. G1 30978(8615) vs. 12795(5496) p =0.0027 IgG B.con.env03 140 CF ExtNDC vs. G2 30978(8615) vs. 19517(9387)p = 0.107 IgG B.con.env03 140 CF ExtNDC vs. G3 30978(8615) vs.16564(6097) p = 0.0047 IgG B.con.env03 140 CF ExtNDC vs. G4 30978(8615)vs. 20783(5844) p = 0.0267 IgG B.con.env03 140 CF ExtNDC vs. G530978(8615) vs. 4440(1466)  p < 0.0001 IgG B.con.env03 140 CF ExtNDC vs.ExtDC 30978(8615) vs. 21725(8919) p = 0.0549 IgG B.con.env03 140 CF G3vs. ExtDC 16564(6097) vs. 21725(8919) p = 0.2137 IgG C.con.env03 140 CFExtNDC vs. G1 52906(8011) vs. 8408(2278)  p < 0.0001 IgG C.con.env03 140CF ExtNDC vs. G2 52906(8011) vs. 11801(4445) p = 0.0002 IgG C.con.env03140 CF ExtNDC vs. G3 52906(8011) vs. 9702(3497)  p < 0.0001 IgGC.con.env03 140 CF ExtNDC vs. G4 52906(8011) vs. 14968(5298) p = 0.0002IgG C.con.env03 140 CF ExtNDC vs. G5 52906(8011) vs. 4996(2451)  p =0.0001 IgG C.con.env03 140 CF ExtNDC vs. ExtDC 52906(8011) vs.31053(7462) p = 0.0019 IgG C.con.env03 140 CF G3 vs. ExtDC  9702(3497)vs. 31053(7462) p = 0.0001 IgG Con S gp140 CFI ExtNDC vs. G1 48885(7813)vs. 17947(4889) p = 0.0001 IgG Con S gp140 CFI ExtNDC vs. G2 48885(7813)vs. 23372(7345) p = 0.0027 IgG Con S gp140 CFI ExtNDC vs. G3 48885(7813)vs. 22509(6103) p = 0.0002 IgG Con S gp140 CFI ExtNDC vs. G4 48885(7813)vs. 27231(8472) p = 0.0027 IgG Con S gp140 CFI ExtNDC vs. G5 48885(7813)vs. 11026(4791) p = 0.0004 IgG Con S gp140 CFI ExtNDC vs. ExtDC 48885(7813) vs. 42254(10504) p = 0.2161 IgG Con S gp140 CFI G3 vs.ExtDC  22510(6103) vs. 42254(10504) p = 0.0018 IgG gp70.B.CaseA V1.V2ExtNDC vs. G1 43424(7370) vs. 2764(1709)  p < 0.0001 IgG gp70.B.CaseAV1.V2 ExtNDC vs. G2 43424(7370) vs. 10488(4754) p = 0.0004 IgGgp70.B.CaseA V1.V2 ExtNDC vs. G3 43424(7370) vs. 5563(3734)  p = 0.0008IgG gp70.B.CaseA V1.V2 ExtNDC vs. G4 43424(7370) vs. 8720(4527)  p =0.0036 IgG gp70.B.CaseA V1.V2 ExtNDC vs. G5 43424(7370) vs. 1591(2327) p = 0.0068 IgG gp70.B.CaseA V1.V2 ExtNDC vs. ExtDC 43424(7370) vs.10166(5334) p = 0.0019 IgG gp70.B.CaseA V1.V2 G3 vs. ExtDC  5563(3734)vs. 10166(5334) p = 0.149 IgA 00MSA 4076 gp140 ExtNDC vs. G1 134(135)vs. 10(3)   p = 0.0002 IgA 00MSA 4076 gp140 ExtNDC vs. G2 134(135) vs.9(3)   p < 0.0001 IgA 00MSA 4076 gp140 ExtNDC vs. G3 134(135) vs.13(5)   p = 0.0003 IgA 00MSA 4076 gp140 ExtNDC vs. G4 134(135) vs.21(31)  p = 0.0036 IgA 00MSA 4076 gp140 ExtNDC vs. G5 134(135) vs.13(3)   p = 0.0005 IgA 00MSA 4076 gp140 ExtNDC vs. ExtDC 134(135) vs.261(202) p = 0.1233 IgA 00MSA 4076 gp140 G3 vs. ExtDC   13(5) vs.261(202) p = 0.0001 IgA A1.con.env03 140 CF ExtNDC vs. G1 312(614) vs.9(5)   p = 0.002 IgA A1.con.env03 140 CF ExtNDC vs. G2 312(614) vs.8(6)   p = 0.0017 IgA A1.con.env03 140 CF ExtNDC vs. G3 312(614) vs.16(5)   p = 0.0107 IgA A1.con.env03 140 CF ExtNDC vs. G4 312(614) vs.42(49)  p = 0.0788 IgA A1.con.env03 140 CF ExtNDC vs. G5 312(614) vs.12(8)   p = 0.0038 IgA A1.con.env03 140 CF ExtNDC vs. ExtDC 312(614) vs.313(577) p = 0.9877 IgA A1.con.env03 140 CF G3 vs. ExtDC   16(5) vs.313(577) p = 0.0391 IgA B.con.env03 140 CF ExtNDC vs. G1 125(94) vs.10(3)  p < 0.0001 IgA B.con.env03 140 CF ExtNDC vs. G2 125(94) vs.9(4)   p < 0.0001 IgA B.con.env03 140 CF ExtNDC vs. G3 125(94) vs.11(4)  p < 0.0001 IgA B.con.env03 140 CF ExtNDC vs. G4 125(94) vs.25(24)  p = 0.0037 IgA B.con.env03 140 CF ExtNDC vs. G5 125(94) vs.10(4)  p < 0.0001 IgA B.con.env03 140 CF ExtNDC vs. ExtDC  125(94) vs.454(316) p = 0.0065 IgA B.con.env03 140 CF G3 vs. ExtDC   11(4) vs.454(316) p < 0.0001 IgA C.con.env03 140 CF ExtNDC vs. G1 427(683) vs.29(19)  p = 0.0033 IgA C.con.env03 140 CF ExtNDC vs. G2 427(683) vs.17(8)   p = 0.0007 IgA C.con.env03 140 CF ExtNDC vs. G3 427(683) vs.38(25)  p = 0.008 IgA C.con.env03 140 CF ExtNDC vs. G4 427(683) vs.60(81)  p = 0.0255 IgA C.con.env03 140 CF ExtNDC vs. G5 427(683) vs.20(15)  p = 0.0011 IgA C.con.env03 140 CF ExtNDC vs. ExtDC 427(683) vs.450(448) p = 0.8141 IgA C.con.env03 140 CF G3 vs. ExtDC  38(25) vs.450(448) p = 0.0271 IgA Con S gp140 CFI ExtNDC vs. G1 390(779) vs.18(17)  p = 0.0032 IgA Con S gp140 CFI ExtNDC vs. G2 390(779) vs.16(8)   p = 0.0024 IgA Con S gp140 CFI ExtNDC vs. G3 390(779) vs.24(11)  p = 0.008 IgA Con S gp140 CFI ExtNDC vs. G4 390(779) vs.111(197) p = 0.1235 IgA Con S gp140 CFI ExtNDC vs. G5 390(779) vs.13(13)  p = 0.0012 IgA Con S gp140 CFI ExtNDC vs. ExtDC  390(779) vs.850(1136) p = 0.3007 IgA Con S gp140 CFI G3 vs. ExtDC   24(11) vs.850(1136) p = 0.0078 IgA gp70.B.CaseA V1.V2 ExtNDC vs. G1 64(57) vs.9(7)  p = 0.0019 IgA gp70.B.CaseA V1.V2 ExtNDC vs. G2 64(57) vs. 13(9) p = 0.0136 IgA gp70.B.CaseA V1.V2 ExtNDC vs. G3 64(57) vs. 28(43) p =0.0831 IgA gp70.B.CaseA V1.V2 ExtNDC vs. G4 64(57) vs. 22(22) p = 0.0679IgA gp70.B.CaseA V1.V2 ExtNDC vs. G5 64(57) vs. 7(5)  p = 0.0027 IgAgp70.B.CaseA V1.V2 ExtNDC vs. ExtDC 64(57) vs. 48(34) p = 0.6556 IgAgp70.B.CaseA V1.V2 G3 vs. ExtDC 28(43) vs. 48(34) p = 0.1912 *p valuesare from t-test assuming unequal variance between groups.

TABLE 7 Peak time point neutralization ID50 titers pair-wise comparisonof non-DC targeting ExtNDC-N2[NP]2 against DC-targeted groups (G1 to G5in the current study and ExtDC- N2Lp3) as well as ExtDC against G3.Assay:Isolate Comparison Mean (SD) p Value* A3R5:TV1.21.LucR.T2A.ectoExtNDC vs. G1 103.87(64.25) vs. 18.17(9.52)  p = 0.0002A3R5:TV1.21.LucR.T2A.ecto ExtNDC vs. G2 103.87(64.25) vs. 26.83(14.11) p = 0.0037 A3R5:TV1.21.LucR.T2A.ecto ExtNDC vs. G3 103.87(64.25) vs.12.67(6.53)  p < 0.0001 A3R5:TV1.21.LucR.T2A.ecto ExtNDC vs. G4103.87(64.25) vs. 32.67(29.47)  p = 0.0173 A3R5:TV1.21.LucR.T2A.ectoExtNDC vs. G5 103.87(64.25) vs. 17.5(11.62)  p = 0.0003A3R5:TV1.21.LucR.T2A.ecto ExtNDC vs. ExtDC 103.87(64.25) vs.49.5(12.06)  p = 0.0531 A3R5:TV1.21.LucR.T2A.ecto G3 vs. ExtDC12.67(6.53) vs. 49.5(12.06) p < 0.0001 TZM-bl:MN.3 ExtNDC vs. G160.38(68.86) vs. 14.33(10.61) p = 0.0272 TZM-bl:MN.3 ExtNDC vs. G260.38(68.86) vs. 10(0)    p = 0.0098 TZM-bl:MN.3 ExtNDC vs. G360.38(68.86) vs. 10(0)    p = 0.0098 TZM-bl:MN.3 ExtNDC vs. G460.38(68.86) vs. 10(0)    p = 0.0098 TZM-bl:MN.3 ExtNDC vs. G560.38(68.86) vs. 10(0)    p = 0.0098 TZM-bl:MN.3 ExtNDC vs. ExtDC60.38(68.86) vs. 10(0)    p = 0.0098 TZM-bl:MN.3 G3 vs. ExtDC 10(0) vs.10(0) TZM-bl:MW965.26 ExtNDC vs. G1 3049.38(2653.11) vs. 104.67(62.34)  p < 0.0001 TZM-bl:MW965.26 ExtNDC vs. G2 3049.38(2653.11) vs.248.17(204.56)  p = 0.0006 TZM-bl:MW965.26 ExtNDC vs. G33049.38(2653.11) vs. 110(86.89)     p = 0.0007 TZM-bl:MW965.26 ExtNDCvs. G4 3049.38(2653.11) vs. 453.33(564.57)  p = 0.0026 TZM-bl:MW965.26ExtNDC vs. G5 3049.38(2653.11) vs. 105.67(167.7)   p = 0.0002TZM-bl:MW965.26 ExtNDC vs. ExtDC 3049.38(2653.11) vs. 915.17(898.84)  p= 0.0281 TZM-bl:MW965.26 G3 vs. ExtDC    110(86.89) vs. 915.17(898.84) p= 0.0105 TZM-bl:SF162.LS ExtNDC vs. G1 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS ExtNDC vs. G2 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS ExtNDC vs. G3 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS ExtNDC vs. G4 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS ExtNDC vs. G5 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS ExtNDC vs. ExtDC 92.37(93.54) vs. 10(0)    p =0.0076 TZM-bl:SF162.LS G3 vs. ExtDC 10(0) vs. 10(0) TZM-bl:TH023.6ExtNDC vs. G1 406.5(657.58) vs. 42.5(41.11)  p = 0.0136 TZM-bl:TH023.6ExtNDC vs. G2  406.5(657.58) vs. 108.33(160.32) p = 0.073 TZM-bl:TH023.6ExtNDC vs. G3 406.5(657.58) vs. 98.33(207.2)  p = 0.0301 TZM-bl:TH023.6ExtNDC vs. G4 406.5(657.58) vs. 71.83(75.34)  p = 0.0588 TZM-bl:TH023.6ExtNDC vs. G5 406.5(657.58) vs. 10(0)     p = 0.0005 TZM-bl:TH023.6ExtNDC vs. ExtDC  406.5(657.58) vs. 173.83(228.74) p = 0.3105TZM-bl:TH023.6 G3 vs. ExtDC  98.33(207.2) vs. 173.83(228.74) p = 0.1586*p values are from t-test assuming unequal variance between groups.

TABLE 8 Peak time point ADCC ID50 titers pair-wise comparison of non-DCtargeting ExtNDC-N2[NP]2 against DC-targeted groups (G1 to G5 in thecurrent study and ExtDC N2Lp3) as well as ExtDC against G3. ParameterComparison Mean (SD) p Value* GTL ADCC:peak ExtNDC vs. G1 11.023(5.75)vs. 5.22(6.202)  p = 0.103 GTL ADCC:peak ExtNDC vs. G2 11.023(5.75) vs.6.364(5.121) p = 0.1371 GTL ADCC:peak ExtNDC vs. G3 11.023(5.75) vs.3.81(3.099)  p = 0.0117 GTL ADCC:peak ExtNDC vs. G4 11.023(5.75) vs.8.568(5.331) p = 0.4266 GTL ADCC:peak ExtNDC vs. G5 11.023(5.75) vs.3.469(3.527) p = 0.0107 GTL ADCC:peak ExtNDC vs. ExtDC 11.023(5.75) vs.1.002(0.207) p = 0.0017 GTL ADCC:peak G3 vs. ExtDC  3.81(3.099) vs.1.002(0.207) p = 0.0772 GTL ADCC:auc ExtNDC vs. G1 20.24(9.508) vs.3.063(3.796) p = 0.001 GTL ADCC:auc ExtNDC vs. G2 20.24(9.508) vs.5.908(6.776) p = 0.0064 GTL ADCC:auc ExtNDC vs. G3 20.24(9.508) vs.2.292(1.972) p = 0.0009 GTL ADCC:auc ExtNDC vs. G4 20.24(9.508) vs.6.805(5.879) p = 0.0071 GTL ADCC:auc ExtNDC vs. G5 20.24(9.508) vs.1.678(1.612) p = 0.0008 GTL ADCC:auc ExtNDC vs. ExtDC 20.24(9.508) vs.1.082(0.405) p = 0.0007 GTL ADCC:auc G3 vs. ExtDC 2.292(1.972) vs.1.082(0.405) p = 0.1974 *p values are from t-test assuming unequalvariance between groups

TABLE 9 Peak time point CD4+ and CD8+ T 62 cells HIV-specific cytokinesecreting response pair-wise comparison of non-DC targetingExtNDC-N2[NP]2 against DC-targeted groups (G1 to G5 in the current studyand ExtDC N2Lp3) as well as ExtDC against G3. Parameter Comparison Mean(SD) p Value* CD4+ Any Env AnyCyto ExtNDC vs. G1  0.87(0.514) vs.0.387(0.361) p = 0.0451 CD4+ Any Env AnyCyto ExtNDC vs. G2  0.87(0.514)vs. 0.642(0.343) p = 0.4529 CD4+ Any Env AnyCyto ExtNDC vs. G3 0.87(0.514) vs. 0.305(0.228) p = 0.0184 CD4+ Any Env AnyCyto ExtNDC vs.G4  0.87(0.514) vs. 0.447(0.287) p = 0.0847 CD4+ Any Env AnyCyto ExtNDCvs. G5  0.87(0.514) vs. 0.082(0.023) p < 0.0001 CD4+ Any Env AnyCytoExtNDC vs. ExtDC  0.87(0.514) vs. 0.278(0.146) p = 0.0083 CD4+ Any EnvAnyCyto G3 vs ExtDC 0.305(0.228) vs. 0.278(0.146) p = 0.9744 CD4+ AnyHIV Antigen AnyCyto ExtNDC vs. G1 0.919(0.501) vs. 0.441(0.355) p =0.0555 CD4+ Any HIV Antigen AnyCyto ExtNDC vs. G2 0.919(0.501) vs.0.693(0.359) p = 0.4711 CD4+ Any HIV Antigen AnyCyto ExtNDC vs. G30.919(0.501) vs. 0.338(0.23)  p = 0.0199 CD4+ Any HIV Antigen AnyCytoExtNDC vs. G4 0.919(0.501) vs. 0.471(0.289) p = 0.0784 CD4+ Any HIVAntigen AnyCyto ExtNDC vs. G5 0.919(0.501) vs. 0.115(0.036) p < 0.0001CD4+ Any HIV Antigen AnyCyto ExtNDC vs. ExtDC 0.919(0.501) vs.0.428(0.246) p = 0.0465 CD4+ Any HIV Antigen AnyCyto G3 vs ExtDC 0.338(0.23) vs. 0.428(0.246) p = 0.4299 CD8+ Any Env AnyCyto ExtNDC vs.G1 0.184(0.226) vs. 0.091(0.063) p = 0.3725 CD8+ Any Env AnyCyto ExtNDCvs. G2 0.184(0.226) vs. 0.197(0.169) p = 0.6862 CD8+ Any Env AnyCytoExtNDC vs. G3 0.184(0.226) vs. 0.054(0.01)  p = 0.0634 CD8+ Any EnvAnyCyto ExtNDC vs. G4 0.184(0.226) vs. 0.074(0.033) p = 0.2111 CD8+ AnyEnv AnyCyto ExtNDC vs. G5 0.184(0.226) vs. 0.052(0.006) p = 0.0554 CD8+Any Env AnyCyto ExtNDC vs. ExtDC 0.184(0.226) vs. 0.108(0.056) p =0.6869 CD8+ Any Env AnyCyto G3 vs ExtDC  0.054(0.01) vs. 0.108(0.056) p= 0.0436 CD8+ Any HIV Antigen AnyCyto ExtNDC vs. G1 0.333(0.243) vs.0.148(0.111) p = 0.0462 CD8+ Any HIV Antigen AnyCyto ExtNDC vs. G20.333(0.243) vs. 0.212(0.147) p = 0.2487 CD8+ Any HIV Antigen AnyCytoExtNDC vs. G3 0.333(0.243) vs. 0.071(0.032) p = 0.0003 CD8+ Any HIVAntigen AnyCyto ExtNDC vs. G4 0.333(0.243) vs. 0.139(0.122) p = 0.0399CD8+ Any HIV Antigen AnyCyto ExtNDC vs. G5 0.333(0.243) vs. 0.069(0.045)p = 0.0003 CD8+ Any HIV Antigen AnyCyto ExtNDC vs. ExtDC 0.333(0.243)vs. 0.313(0.227) p = 0.7621 CD8+ Any HIV Antigen AnyCyto G3 vs ExtDC0.071(0.032) vs. 0.313(0.227) p = 0.015 *p values are from t-testassuming unequal variance between groups

TABLE 10 Pair-wise comparison of durability (proportion of change perweek) of binding antibody response among G1-G5 of the current study andExtNDC-N2[NP]2. Analyte Comparison Mean (SD) p Value* 00MSA 4076 gp140ExtNDC vs. G1 −0.075(0.019) vs. −0.118(0.032) p = 0.0343 00MSA 4076gp140 ExtNDC vs. G2 −0.075(0.019) vs. −0.091(0.037) p = 0.3704 00MSA4076 gp140 ExtNDC vs. G3 −0.075(0.019) vs. −0.099(0.016) p = 0.026800MSA 4076 gp140 ExtNDC vs. G4 −0.075(0.019) vs. −0.077(0.035) p =0.9317 00MSA 4076 gp140 ExtNDC vs. G5 −0.075(0.019) vs. −0.108(0.021) p= 0.0119 00MSA 4076 gp140 ExtNDC vs. ExtDC −0.075(0.019) vs.−0.063(0.009) p = 0.129 00MSA 4076 gp140 G1 vs. G2 −0.118(0.032) vs.−0.091(0.037) p = 0.2238 00MSA 4076 gp140 G1 vs. G3 −0.118(0.032) vs.−0.099(0.016) p = 0.2571 00MSA 4076 gp140 G1 vs. G4 −0.118(0.032) vs.−0.077(0.035) p = 0.0691 00MSA 4076 gp140 G1 vs. G5 −0.118(0.032) vs.−0.108(0.021) p = 0.5668 00MSA 4076 gp140 G1 vs. ExtDC −0.118(0.032) vs.−0.063(0.009) p = 0.0151 00MSA 4076 gp140 G2 vs. G3 −0.091(0.037) vs.−0.099(0.016) p = 0.6634 00MSA 4076 gp140 G2 vs. G4 −0.091(0.037) vs.−0.077(0.035) p = 0.5044 00MSA 4076 gp140 G2 vs. G5 −0.091(0.037) vs.−0.108(0.021) p = 0.3546 00MSA 4076 gp140 G2 vs. ExtDC −0.091(0.037) vs.−0.063(0.009) p = 0.1241 00MSA 4076 gp140 G3 vs. G4 −0.099(0.016) vs.−0.077(0.035) p = 0.2052 00MSA 4076 gp140 G3 vs. G5 −0.099(0.016) vs.−0.108(0.021) p = 0.3927 00MSA 4076 gp140 G3 vs. ExtDC −0.099(0.016) vs.−0.063(0.009) p = 0.0014 00MSA 4076 gp140 G4 vs. G5 −0.077(0.035) vs.−0.108(0.021) p = 0.0941 00MSA 4076 gp140 G4 vs. ExtDC −0.077(0.035) vs.−0.063(0.009) p = 0.3884 00MSA 4076 gp140 G5 vs. ExtDC −0.108(0.021) vs.−0.063(0.009) p = 0.002 A1.con.env03 140 CF ExtNDC vs. G1 −0.064(0.016)vs. −0.117(0.016) p < 0.0001 A1.con.env03 140 CF ExtNDC vs. G2−0.064(0.016) vs. −0.09(0.031)  p = 0.1011 A1.con.env03 140 CF ExtNDCvs. G3 −0.064(0.016) vs. −0.095(0.019) p = 0.0088 A1.con.env03 140 CFExtNDC vs. G4 −0.064(0.016) vs. −0.071(0.029) p = 0.6128 A1.con.env03140 CF ExtNDC vs. G5 −0.064(0.016) vs. −0.116(0.02)  p = 0.0004A1.con.env03 140 CF ExtNDC vs. ExtDC −0.064(0.016) vs. −0.059(0.008) p =0.4713 A1.con.env03 140 CF G1 vs. G2 −0.117(0.016) vs. −0.09(0.031)  p =0.1003 A1.con.env03 140 CF G1 vs. G3 −0.117(0.016) vs. −0.095(0.019) p =0.0537 A1.con.env03 140 CF G1 vs. G4 −0.117(0.016) vs. −0.071(0.029) p =0.0095 A1.con.env03 140 CF G1 vs. G5 −0.117(0.016) vs. −0.116(0.02)  p =0.9043 A1.con.env03 140 CF G1 vs. ExtDC −0.117(0.016) vs. −0.059(0.008)p < 0.0001 A1.con.env03 140 CF G2 vs. G3  −0.09(0.031) vs. −0.095(0.019)p = 0.7586 A1.con.env03 140 CF G2 vs. G4  −0.09(0.031) vs. −0.071(0.029)p = 0.292 A1.con.env03 140 CF G2 vs. G5 −0.09(0.031) vs. −0.116(0.02) p= 0.1262 A1.con.env03 140 CF G2 vs. ExtDC  −0.09(0.031) vs.−0.059(0.008) p = 0.0585 A1.con.env03 140 CF G3 vs. G4 −0.095(0.019) vs.−0.071(0.029) p = 0.1241 A1.con.env03 140 CF G3 vs. G5 −0.095(0.019) vs.−0.116(0.02)  p = 0.0906 A1.con.env03 140 CF G3 vs. ExtDC −0.095(0.019)vs. −0.059(0.008) p = 0.0039 A1.con.env03 140 CF G4 vs. G5 −0.071(0.029)vs. −0.116(0.02)  p = 0.0123 A1.con.env03 140 CF G4 vs. ExtDC−0.071(0.029) vs. −0.059(0.008) p = 0.3783 A1.con.env03 140 CF G5 vs.ExtDC  −0.116(0.02) vs. −0.059(0.008) p = 0.0004 B.con.env03 140 CFExtNDC vs. G1 −0.084(0.012) vs. −0.129(0.021) p = 0.0017 B.con.env03 140CF ExtNDC vs. G2 −0.084(0.012) vs. −0.103(0.04)  p = 0.3158 B.con.env03140 CF ExtNDC vs. G3 −0.084(0.012) vs. −0.12(0.013)  p = 0.0004B.con.env03 140 CF ExtNDC vs. G4 −0.084(0.012) vs. −0.1(0.017)  p =0.0895 B.con.env03 140 CF ExtNDC vs. G5 −0.084(0.012) vs. −0.132(0.015)p = 0.0001 B.con.env03 140 CF ExtNDC vs. ExtDC −0.084(0.012) vs.−0.068(0.011) p = 0.023 B.con.env03 140 CF G1 vs. G2 −0.129(0.021) vs.−0.103(0.04)  p = 0.2008 B.con.env03 140 CF G1 vs. G3 −0.129(0.021) vs.−0.12(0.013)  p = 0.3956 B.con.env03 140 CF G1 vs. G4 −0.129(0.021) vs.−0.1(0.017)  p = 0.0268 B.con.env03 140 CF G1 vs. G5 −0.129(0.021) vs.−0.132(0.015) p = 0.7818 B.con.env03 140 CF G1 vs. ExtDC −0.129(0.021)vs. −0.068(0.011) p = 0.0003 B.con.env03 140 CF G2 vs. G3 −0.103(0.04)vs. −0.12(0.013) p = 0.3641 B.con.env03 140 CF G2 vs. G4 −0.103(0.04)vs. −0.1(0.017)  p = 0.8826 B.con.env03 140 CF G2 vs. G5  −0.103(0.04)vs. −0.132(0.015) p = 0.1481 B.con.env03 140 CF G2 vs. ExtDC −0.103(0.04) vs. −0.068(0.011) p = 0.0878 B.con.env03 140 CF G3 vs. G4−0.12(0.013) vs. −0.1(0.017)  p = 0.0538 B.con.env03 140 CF G3 vs. G5 −0.12(0.013) vs. −0.132(0.015) p = 0.1764 B.con.env03 140 CF G3 vs.ExtDC  −0.12(0.013) vs. −0.068(0.011) p < 0.0001 B.con.env03 140 CF G4vs. G5  −0.1(0.017) vs. −0.132(0.015) p = 0.0073 B.con.env03 140 CF G4vs. ExtDC  −0.1(0.017) vs. −0.068(0.011) p = 0.0045 B.con.env03 140 CFG5 vs. ExtDC −0.132(0.015) vs. −0.068(0.011) p < 0.0001 C.con.env03 140CF ExtNDC vs. G1 −0.058(0.016) vs. −0.091(0.028) p = 0.035 C.con.env03140 CF ExtNDC vs. G2 −0.058(0.016) vs. −0.045(0.067) p = 0.6572C.con.env03 140 CF ExtNDC vs. G3 −0.058(0.016) vs. −0.069(0.029) p =0.4269 C.con.env03 140 CF ExtNDC vs. G4 −0.058(0.016) vs. −0.041(0.014)p = 0.0646 C.con.env03 140 CF ExtNDC vs. G5 −0.058(0.016) vs.−0.092(0.035) p = 0.0625 C.con.env03 140 CF ExtNDC vs. ExtDC−0.058(0.016) vs. −0.062(0.007) p = 0.5735 C.con.env03 140 CF G1 vs. G2−0.091(0.028) vs. −0.045(0.067) p = 0.1682 C.con.env03 140 CF G1 vs. G3−0.091(0.028) vs. −0.069(0.029) p = 0.2139 C.con.env03 140 CF G1 vs. G4−0.091(0.028) vs. −0.041(0.014) p = 0.0056 C.con.env03 140 CF G1 vs. G5−0.091(0.028) vs. −0.092(0.035) p = 0.9297 C.con.env03 140 CF G1 vs.ExtDC −0.091(0.028) vs. −0.062(0.007) p = 0.0511 C.con.env03 140 CF G2vs. G3 −0.045(0.067) vs. −0.069(0.029) p = 0.4468 C.con.env03 140 CF G2vs. G4 −0.045(0.067) vs. −0.041(0.014) p = 0.9069 C.con.env03 140 CF G2vs. G5 −0.045(0.067) vs. −0.092(0.035) p = 0.1649 C.con.env03 140 CF G2vs. ExtDC −0.045(0.067) vs. −0.062(0.007) p = 0.5685 C.con.env03 140 CFG3 vs. G4 −0.069(0.029) vs. −0.041(0.014) p = 0.0734 C.con.env03 140 CFG3 vs. G5 −0.069(0.029) vs. −0.092(0.035) p = 0.2355 C.con.env03 140 CFG3 vs. ExtDC −0.069(0.029) vs. −0.062(0.007) p = 0.5723 C.con.env03 140CF G4 vs. G5 −0.041(0.014) vs. −0.092(0.035) p = 0.0142 C.con.env03 140CF G4 vs. ExtDC −0.041(0.014) vs. −0.062(0.007) p = 0.0172 C.con.env03140 CF G5 vs. ExtDC −0.092(0.035) vs. −0.062(0.007) p = 0.0848 Con Sgp140 CFI ExtNDC vs. G1 −0.055(0.017) vs. −0.097(0.025) p = 0.0068 Con Sgp140 CFI ExtNDC vs. G2 −0.055(0.017) vs. −0.057(0.061) p = 0.9456 Con Sgp140 CFI ExtNDC vs. G3 −0.055(0.017) vs. −0.075(0.024) p = 0.1181 Con Sgp140 CFI ExtNDC vs. G4 −0.055(0.017) vs. −0.052(0.031) p = 0.8115 Con Sgp140 CFI ExtNDC vs. G5 −0.055(0.017) vs. −0.108(0.028) p = 0.0034 Con Sgp140 CFI ExtNDC vs. ExtDC −0.055(0.017) vs. −0.052(0.01)  p = 0.7156Con S gp140 CFI G1 vs. G2 −0.097(0.025) vs. −0.057(0.061) p = 0.1803 ConS gp140 CFI G1 vs. G3 −0.097(0.025) vs. −0.075(0.024) p = 0.1527 Con Sgp140 CFI G1 vs. G4 −0.097(0.025) vs. −0.052(0.031) p = 0.0182 Con Sgp140 CFI G1 vs. G5 −0.097(0.025) vs. −0.108(0.028) p = 0.4876 Con Sgp140 CFI G1 vs. ExtDC −0.097(0.025) vs. −0.052(0.01)  p = 0.0053 Con Sgp140 CFI G2 vs. G3 −0.057(0.061) vs. −0.075(0.024) p = 0.5186 Con Sgp140 CFI G2 vs. G4 −0.057(0.061) vs. −0.052(0.031) p = 0.8562 Con Sgp140 CFI G2 vs. G5 −0.057(0.061) vs. −0.108(0.028) p = 0.1033 Con Sgp140 CFI G2 vs. ExtDC −0.057(0.061) vs. −0.052(0.01)  p = 0.8649 Con Sgp140 CFI G3 vs. G4 −0.075(0.024) vs. −0.052(0.031) p = 0.1712 Con Sgp140 CFI G3 vs. G5 −0.075(0.024) vs. −0.108(0.028) p = 0.0536 Con Sgp140 CFI G3 vs. ExtDC −0.075(0.024) vs. −0.052(0.01)  p = 0.0737 Con Sgp140 CFI G4 vs. G5 −0.052(0.031) vs. −0.108(0.028) p = 0.0072 Con Sgp140 CFI G4 vs. ExtDC −0.052(0.031) vs. −0.052(0.01)  p = 0.9582 Con Sgp140 CFI G5 vs. ExtDC −0.108(0.028) vs. −0.052(0.01)  p = 0.0031gp70.B.CaseA V1.V2 ExtNDC vs. G1 −0.076(0.012) vs. −0.139(0.036) p =0.0069 gp70.B.CaseA V1.V2 ExtNDC vs. G2 −0.076(0.012) vs. −0.107(0.034)p = 0.0725 gp70.B.CaseA V1.V2 ExtNDC vs. G3 −0.076(0.012) vs.−0.125(0.029) p = 0.007 gp70.B.CaseA V1.V2 ExtNDC vs. G4 −0.076(0.012)vs. −0.064(0.081) p = 0.7421 gp70.B.CaseA V1.V2 ExtNDC vs. G5−0.076(0.012) vs. −0.142(0.041) p = 0.1063 gp70.B.CaseA V1.V2 ExtNDC vs.ExtDC −0.076(0.012) vs. −0.085(0.011) p = 0.1501 gp70.B.CaseA V1.V2 G1vs. G2 −0.139(0.036) vs. −0.107(0.034) p = 0.1533 gp70.B.CaseA V1.V2 G1vs. G3 −0.139(0.036) vs. −0.125(0.029) p = 0.4923 gp70.B.CaseA V1.V2 G1vs. G4 −0.139(0.036) vs. −0.064(0.081) p = 0.081 gp70.B.CaseA V1.V2 G1vs. G5 −0.139(0.036) vs. −0.142(0.041) p = 0.9146 gp70.B.CaseA V1.V2 G1vs. ExtDC −0.139(0.036) vs. −0.085(0.011) p = 0.014 gp70.B.CaseA V1.V2G2 vs. G3 −0.107(0.034) vs. −0.125(0.029) p = 0.3521 gp70.B.CaseA V1.V2G2 vs. G4 −0.107(0.034) vs. −0.064(0.081) p = 0.2721 gp70.B.CaseA V1.V2G2 vs. G5 −0.107(0.034) vs. −0.142(0.041) p = 0.2918 gp70.B.CaseA V1.V2G2 vs. ExtDC −0.107(0.034) vs. −0.085(0.011) p = 0.1795 gp70.B.CaseAV1.V2 G3 vs. G4 −0.125(0.029) vs. −0.064(0.081) p = 0.1336 gp70.B.CaseAV1.V2 G3 vs. G5 −0.125(0.029) vs. −0.142(0.041) p = 0.5754 gp70.B.CaseAV1.V2 G3 vs. ExtDC −0.125(0.029) vs. −0.085(0.011) p = 0.0183gp70.B.CaseA V1.V2 G4 vs. G5 −0.064(0.081) vs. −0.142(0.041) p = 0.101gp70.B.CaseA V1.V2 G4 vs. ExtDC −0.064(0.081) vs. −0.085(0.011) p =0.5557 gp70.B.CaseA V1.V2 G5 vs. ExtDC −0.142(0.041) vs. −0.085(0.011) p= 0.1386 *p values are from t-test assuming unequal variance betweengroups

TABLE 11 Pair-wise comparison of durability (proportion of change perweek) of neutralizing activity among G1-G5 of the current study andExtNDC-N2[NP]2. Parameter Comparison Mean (SD) p Value* TZM-bl:MW965.26ExtNDC vs. G1 −0.09(0.006) vs. −0.14(0.007) p < 0.0001 TZM-bl:MW965.26ExtNDC vs. G2  −0.09(0.006) vs. −0.116(0.054) p = 0.3022 TZM-bl:MW965.26ExtNDC vs. G3  −0.09(0.006) vs. −0.136(0.031) p = 0.0586 TZM-bl:MW965.26ExtNDC vs. G4  −0.09(0.006) vs. −0.124(0.026) p = 0.0239 TZM-bl:MW965.26ExtNDC vs. G5  −0.09(0.006) vs. −0.122(0.018) p = 0.017 TZM-bl:MW965.26ExtNDC vs. ExtDC  −0.09(0.006) vs. −0.078(0.011) p = 0.0473TZM-bl:MW965.26 G1 vs. G2  −0.14(0.007) vs. −0.116(0.054) p = 0.3268TZM-bl:MW965.26 G1 vs. G3  −0.14(0.007) vs. −0.136(0.031) p = 0.7903TZM-bl:MW965.26 G1 vs. G4  −0.14(0.007) vs. −0.124(0.026) p = 0.1843TZM-bl:MW965.26 G1 vs. G5  −0.14(0.007) vs. −0.122(0.018) p = 0.0875TZM-bl:MW965.26 G1 vs. ExtDC  −0.14(0.007) vs. −0.078(0.011) p < 0.0001TZM-bl:MW965.26 G2 vs. G3 −0.116(0.054) vs. −0.136(0.031) p = 0.486TZM-bl:MW965.26 G2 vs. G4 −0.116(0.054) vs. −0.124(0.026) p = 0.7603TZM-bl:MW965.26 G2 vs. G5 −0.116(0.054) vs. −0.122(0.018) p = 0.8167TZM-bl:MW965.26 G2 vs. ExtDC −0.116(0.054) vs. −0.078(0.011) p = 0.1522TZM-bl:MW965.26 G3 vs. G4 −0.136(0.031) vs. −0.124(0.026) p = 0.5462TZM-bl:MW965.26 G3 vs. G5 −0.136(0.031) vs. −0.122(0.018) p = 0.4605TZM-bl:MW965.26 G3 vs. ExtDC −0.136(0.031) vs. −0.078(0.011) p = 0.0282TZM-bl:MW965.26 G4 vs. G5 −0.124(0.026) vs. −0.122(0.018) p = 0.8805TZM-bl:MW965.26 G4 vs. ExtDC −0.124(0.026) vs. −0.078(0.011) p = 0.0057TZM-bl:MW965.26 G5 vs. ExtDC −0.122(0.018) vs. −0.078(0.011) p = 0.0031*p values are from t-test assuming unequal variance between groups.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An anti-Dendritic Cell (DC) receptor antibody-HIV antigen fusionprotein comprising, (i) an anti-DC receptor heavy chain (HDCR)-HIVantigen (Ag) fusion protein comprising the formula: HDCR-Ag, wherein Agis a polypeptide with at least 80% sequence identity to SEQ ID NO:1; and(ii) an anti-DC receptor light chain (LDCR).
 2. The fusion protein ofclaim 1, wherein the fusion protein further comprises one or morepeptide linkers (PL).
 3. The fusion protein of claim 2, wherein thefusion protein comprises: (i) HDCR-PL-Ag; and (ii) LDCR.
 4. The fusionprotein of claim 1, further comprising one or more joining sites (JS),wherein the joining site comprises alanine and serine residues.
 5. Thefusion protein of claim 1, further comprising one or more joining sites(JS), wherein the joining site consists of alanine and serine residues.6. The fusion protein of claim 1, wherein the fusion protein comprises:(i) HDCR-JS-PL-JS-Ag-JS; and (ii) LCD40; wherein JS is a joining siteand PL is a peptide linker, and LCD40 is an anti-CD40 light chain. 7.The fusion protein of claim 2, wherein the peptide linker comprises apolypeptide with at least 80% sequence identity to SEQ ID NO:2.
 8. Thefusion protein of claim 3, wherein PL-Ag comprises a polypeptide with atleast 80% identity to SEQ ID NO:3.
 9. The fusion protein of claim 1,which is an anti-CD40 antibody-HIV antigen fusion protein comprising (i)an anti-CD40 heavy chain (HCD40)-HIV antigen (Ag) fusion proteincomprising the formula: HCD40-Ag, wherein Ag is a polypeptide with atleast 80% sequence identity to SEQ ID NO:1; and (ii) an anti-CD40 lightchain (LCD40).
 10. The fusion protein of claim 9, wherein the anti-CD40antibody is a human or humanized anti-CD40 antibody.
 11. The fusionprotein of claim 9, wherein the anti-CD40 antibody comprises human IgG4heavy chain constant region.
 12. The fusion protein of claim 11, whereinthe human IgG4 heavy chain constant region comprises one or both ofS241P and L248E substitutions.
 13. The fusion protein of claim 9,wherein the HCD40 comprises a polypeptide with at least 80% sequenceidentity to SEQ ID NO:4.
 14. The fusion protein of claim 9, wherein theLCD40 comprises a polypeptide with at least 80% sequence identity to SEQID NO:5.
 15. The fusion protein of claim 9, wherein HCDR comprises thecomplementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1Hhaving the amino acid sequence GFTFSDYYMY (SEQ ID NO:10), the CDR2Hhaving the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:11), andthe CDR3H having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:12). 16.The fusion protein of claim 9, wherein LCD40 comprises thecomplementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1Lhaving the amino acid sequence SASQGISNYLN (SEQ ID NO.:13) the CDR2Lhaving the amino acid sequence YTSILHS (SEQ ID NO.:14) and the CDR3Lhaving the amino acid sequence QQFNKLPPT (SEQ ID NO.:15).
 17. The fusionprotein of claim 9, comprising (i) a HCD40-Ag fusion protein comprisingan amino acid sequence with at least 80% sequence identity to SEQ IDNO:6; and (ii) a LCD40 comprising an amino acid sequence with at least80% identity to SEQ ID NO:5.
 18. The fusion protein of claim 9, whereinHCDR comprises the complementarity determining regions CDR1H, CDR2H andCDR3H, the CDR1H having the amino acid sequence GYSFTGYYMH (SEQ IDNO.:18), the CDR2H having the amino acid sequence RINPYNGATSYNQNFKD (SEQID NO.:19), and the CDR3H having the amino acid sequence EDYVY (SEQ IDNO.:20).
 19. The fusion protein of claim 9, wherein LCD40 comprises thecomplementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1Lhaving the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO.:21) theCDR2L having the amino acid sequence KVSNRFS (SEQ ID NO.:22) and theCDR3L having the amino acid sequence SQSTHVPWT (SEQ ID NO.:23).
 20. Thefusion protein of claim 9, wherein HCDR comprises the complementaritydetermining regions CDR1H, CDR2H and CDR3H, the CDR1H having the aminoacid sequence GYTFTDYVLH (SEQ ID NO.:26), the CDR2H having the aminoacid sequence YINPYNDGTKYNEKFKG (SEQ ID NO.:27), and the CDR3H havingthe amino acid sequence GYPAYSGYAMDY (SEQ ID NO.:28).
 21. The fusionprotein of claim 9, wherein LCD40 comprises the complementaritydetermining regions CDR1L, CDR2L and CDR3L, the CDR1L having the aminoacid sequence RASQDISNYLN (SEQ ID NO.:29) the CDR2L having the aminoacid sequence YTSRLHS (SEQ ID NO.:30) and the CDR3L having the aminoacid sequence HHGNTLPWT (SEQ ID NO.:31). 22.-33. (canceled)
 34. Avaccine composition comprising the fusion protein of claim 1 and apharmaceutically acceptable vehicle.
 35. A method for treating orpreventing HIV in a subject, the method comprising administering thefusion protein of claim
 1. 36. A method for enhancing a T cell responseand/or antibody cell response in a subject, the method comprisingadministering the fusion protein of claim
 1. 37. A method for inducingIgG binding antibody responses to V1V2 region antigens in a subject inneed thereof, the method comprising administering the fusion protein ofclaim
 1. 38.-49. (canceled)